Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials
Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials
Bradley M. WertheimID 0 1
Yi-Dong Lin 1
Ying-Yi Zhang 1
Andriy O. Samokhin 1
George A. Alba 1
Elena Arons 1
Paul B. Yu 1
Bradley A. Maron 1
0 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital , Boston, MA , United States of America, 2 Department of Medicine, Division of Cardiovascular Medicine, Brigham and Women's Hospital , Boston, MA , United States of America, 3 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital , Boston, MA , United States of America
1 Editor: James West, Vanderbilt University Medical Center , UNITED STATES
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Transcriptomic analysis of pulmonary microvascular endothelial cells from experimental
models offers insight into pulmonary arterial hypertension (PAH) pathobiology. However,
culturing may alter the molecular profile of endothelial cells prior to analysis, limiting the
translational relevance of results. Here we present a novel and validated method for
isolating RNA from pulmonary microvascular endothelial cells (PMVECs) ex vivo that does not
require cell culturing. Initially, presumed rat PMVECs were isolated from rat peripheral lung
tissue using tissue dissociation and enzymatic digestion, and cells were cultured until
confluence to assess endothelial marker expression. Anti-CD31, anti-von Willebrand Factor, and
anti-?-smooth muscle actin immunocytochemistry/immunofluorescence signal was
detected in presumed rat PMVECs, but also in non-endothelial cell type controls. By
contrast, flow cytometry using an anti-CD31 antibody and isolectin 1-B4 (from Griffonia
simplicifolia) was highly specific for rat PMVECs. We next developed a strategy in which the
addition of an immunomagnetic selection step for CD31+ cells permitted culture-free
isolation of rat PMVECs ex vivo for RNA isolation and transcriptomic analysis using
fluorescence-activated cell sorting. Heterogeneity in the validity and reproducibility of results using
commercial antibodies against endothelial surface markers corresponded to a substantial
burden on laboratory time, labor, and scientific budget. We demonstrate a novel protocol for
the culture-free isolation and transcriptomic analysis of rat PMVECs with translational
relevance to PAH. In doing so, we highlight wide variability in the quality of commonly used
biological reagents, which emphasizes the importance of investigator-initiated validation of
Foundation, Cardiovascular Medicine Educational
Research Foundation [CMREF]). The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
Competing interests: The authors have declared
that no competing interests exist.
Abbreviations: a.u., arbitrary units; Ab, antibody;
AF 488, Alexa Fluor 488; AF 647, Alexa Fluor 647;
DAB, 3, 3?-diaminobenzidine; GS-IB4, isolectin 1-B4
from Griffonia simplicifolia; HLF, human lung
fibroblast; HPAEC, human pulmonary artery
endothelial cell; HPASMC, human pulmonary artery
smooth muscle cell; ICC, immunocytochemistry;
IF, immunofluorescence; PAH, pulmonary arterial
hypertension; PE, phycoerythrin; PMVEC, rat
pulmonary microvascular endothelial cell; MCT,
monocrotaline; RECA-1, rat endothelial cell
antigen-1; RIN, RNA integrity number; RLF, rat
lung fibroblast; RPAEC, rat pulmonary artery
endothelial cell; RPASMC, rat pulmonary artery
smooth muscle cell; S.E., standard error; vWF, von
Willebrand factor; ?-SMA, ?-smooth muscle actin.
Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by
dysregulated transcriptional mechanisms that promote endothelial dysfunction [
pulmonary artery endothelial cells (PAECs) from PAH patients is optimal, but access is limited,
in part, by low disease prevalence and technical obstacles [
]. Therefore, studying PAECs
from PAH animal models offers an important and well-established alternative approach to
analyzing disease-specific pathobiological mechanisms . Protocols for isolating primary PAECs
from PAH models have been reported previously, but these strategies require passaging cells in
vitro to ensure a sufficient population for further analysis [
]. However, sequential passaging
may alter the phenotype and molecular program of cells [
]. Effective cell isolation without
serial passaging is possible,[
] but has not been reported for rodent PAECs.
Limited reproducibility of published scientific results has led to an emerging initiative
among funding sponsors, including the National Institutes of Health, that emphasizes data
]. The widespread availability of commercial biomedical products has simplified
reagent preparation and improved laboratory efficiency. However, inconsistent product
quality?for example, uncertain binding epitopes among some commercial antibodies?may
contribute to variability in experimental biology. In turn, data validating these biotechnologies is
likely to improve rigor of scientific findings, but is rarely reported independently by
Microvascular endothelial dysfunction has been implicated in multiple aspects of PAH
pathobiology including angiogenesis, proliferation, apoptosis, and adaptation to shear stress
]. In this report, we describe a practical method for isolating high-quality mRNA for
transcriptomic analysis from rat pulmonary microvascular endothelial cells (PMVECs) ex vivo
without cell passaging. In doing so, we also demonstrate wide variability in the quality of
purchased laboratory reagents. Together, the current work outlines a cell culture-free approach to
studying PMVECs, and reinforces the use of commercial biomaterials without on-site
validation as a modifiable step toward enhancing the reproducibility of data in PAH.
Methods and results
Cell culture and reagents
Human pulmonary artery endothelial cells (HPAECs), human lung fibroblasts (HLFs), and
human pulmonary artery smooth muscle cells (HPASMCs) were purchased from Lonza. Rat
pulmonary artery endothelial cells (RPAECs), rat pulmonary artery smooth muscle cells
(RPASMCs), and rat lung fibroblasts (RLFs) were purchased from Cell Biologics. The details
of each cell type are provided in the S1 Supporting Information. Rat PMVECs were harvested
in our laboratory from the peripheral region of the lung, expressed CD31 (Santa Cruz 376764
[Ab #1] for immunofluorescence and immunohistochemistry; R & D Biosystems FAB3628P
[Ab #20] for flow cytometry), and, in some experiments were confirmed by co-labeling with
isolectin 1-B4 from Griffonia simplicifolia (GS-IB4) (Thermo Fisher, Catalog #I21411). Cell
culture was performed under standard conditions (37?C, 5% CO2, 90% humidity) in
vendorrecommended media (S1 Supporting Information). Cell passages 1?8 were used for
experiments. Details on commercially purchased antibodies (Abs #1?22) used for experiments are
presented in Table 1.
All statistical analyses were performed using Origin Pro 2015 version b9.2.272. The unpaired
Student?s t-test was used for experiments involving comparisons between two samples and
2 / 18
3 / 18
aa, amino acid; AF 488, Alexa Fluor 488; AF 647, Alexa Fluor 647; APC, allophycocyanin; C, chicken; Co, cow; D, dog; FC, flow cytometry; G, goat; GP, guinea pig; H,
human; Ho, horse; IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; M, mouse; NHP, non-human primate; P, pig; PE, phycoerytherin;
R, rat; Rab, rabbit; RM, rhesus monkey; S, sheep; WB, western blot.
one-way analysis of variance (ANOVA) was used for comparisons involving three or more
samples. Post-hoc analyses were performed using the method of Tukey. Data are presented as
mean ? SE. P < 0.05 was used to define statistical significance. Initial data review of
immunocytochemistry, immunofluorescence, and flow cytometry experiments was performed by a
blinded investigator whenever possible. At least three technical or biological replicates were
used per experiment. For colocalization, data were measured in at least 3 cells/field in at least 3
randomly-selected fields/technical replicate.
Cell isolation from experimental PAH in vivo
The overarching objective of this project was to isolate high-quality mRNA from rat
PMVECs acquired ex vivo without tissue culturing. We chose to study rats based on severe
histopathological remodeling and pulmonary hypertension that are reported for
experimental PAH in this species compared to other rodent models [
]. Animals were handled in
accordance with the National Institutes of Health Guide for the Care and Use of Laboratory
Animals and all procedures were approved by the Brigham and Women?s Hospital Animal
Care and Use Committee. A summary of the overall experimental workflow is provided in
We first focused on immunophenotyping cells harvested from normal rats to demonstrate
endothelial cell type (Fig 1A). Male Sprague Dawley rats (200-225g, Charles River) were
anesthetized with ketamine (50 mg/kg)/xylazine (10 mg/kg), sacrificed by exsanguination under
general anesthesia, and the outer ~4 mm of peripheral lung tissue was resected [
whole lung segments were stored in cold Dulbecco?s Modified Eagle Medium (Life
Technologies, catalog #11995?065) briefly until digestion was performed by submerging the tissue in
type II collagenase (1 mg/mL, Worthington Biochemical, catalog #LS004176) for 20 min at
37?C, with 5% CO2 and 90% humidity.
Next, the digested lung fragments were transferred to a culture dish containing endothelial
cell-specific culture medium (Vasculife with EnGS Life Factors Kit, Lifeline Cell Technology,
catalog #LL-0004) supplemented with 1% (vol/vol) penicillin/streptomycin (Thermo Fisher,
catalog #15140122)/amphotericin B (catalog #A2942-50ML), minced with sterile scissors
(~100 times), and the tissue homogenate was filtered through a 70 ?m cell strainer (Falcon,
catalog #352350). The filtrate was then passed through a 20 ?m strainer (Pleuriselect, catalog
#43-50020-01). Trapped cells were eluted into culture medium and centrifuged at 330 x g for 5
min to generate a cell pellet. The pellet was resuspended and plated on a gelatin-coated P60
4 / 18
Fig 1. Overall approach to the isolation and phenotyping of rat pulmonary microvascular endothelial cells (PMVECs). (A) Flow diagram of the initial strategy for
presumed rat PMVEC isolation and phenotyping by immunocytochemistry and immunofluorescence. (B) Approach to isolating and confirming rat PMVECs acquired
ex vivo by fluorescence activated cell sorting. The figure corresponding to data for a step in the approach is provided. HLF, human lung fibroblast; HPAEC, human
pulmonary artery endothelial cell; HPASMC, human pulmonary artery smooth muscle cell; MCT, monocrotaline; RLF, rat lung fibroblast; RPAEC, rat pulmonary
artery endothelial cell; RPASMC, rat pulmonary artery smooth muscle cell; rat PMVEC, rat pulmonary microvascular endothelial cell; SD, Sprague Dawley.
culture dish, incubated under standard conditions in endothelial-selective culture medium
(Vasculife with EnGS Life Factors Kit, Lifeline Cell Technology) supplemented with 1% (vol./
vol.) penicillin/streptomycin. Cells were grown to confluence with culture medium changes
every 48 hr.
An endothelial phenotype was confirmed in confluent cells by phase contrast microscopy.
These were presumed to be rat PMVECs (referred to as ?presumed rat PMVECs? throughout).
Rat PMVECs used in ICC, IF, and pilot flow cytometry experiments were cultured (Fig 1A),
and rat PMVECs used for RNA isolation were not cultured (Fig 1B).
5 / 18
Determining cell type by immunocytochemistry
Immunocytochemistry (ICC) was performed on presumed rat PMVECs using the
3,3?-diaminobenzidine substrate (DAB) method, as reported by our laboratory previously, with some
]. In addition, we confirmed with the vendors that the tested antibodies were
compatible with this method. Cells were fixed in ice cold acetone or methanol, blocked in 10%
bovine serum albumin (BSA), and then incubated with anti-CD31 primary antibodies #1 or
#2, or anti-IgG (Ab #3) as control (dilution 1:100). The secondary antibodies (Abs #4,5) were
incubated at a dilution 1:500. Cells were imaged using an Olympus BX51 microscope, Retiga
3000 camera (1?5 randomly-selected fields/condition). The DAB substrate luminosity was
quantified using Image J (NIH) and expressed in arbitrary units (a.u.) normalized to IgG.
There was no meaningful difference in CD31 or ?-SMA signal between acetone and methanol
fixation (S1 Fig).
We observed that CD31 expression varied significantly between Ab #1 and Ab #2 (3.6 ? 0.2
vs. 0.7 ? 0.2 a.u., p = 5.7 x 10?4, N = 3/condition) (Fig 2A). Based on this result, we tested the
specificity of anti-CD31 ICC using Ab #1 (which had the stronger signal of the two anti-CD31
Abs) in HPASMCs, HLFs, and presumed rat PMVECs. We also performed ICC using an
antibody against von Willebrand factor (vWF) (Ab #6) and anti-?-smooth muscle actin (?-SMA)
(Ab #7) (dilution 1:100?1:1000), which are commonly used endothelial- and smooth muscle
cell-specific markers, respectively. We observed CD31 expression in presumed rat PMVECs,
but it was not significantly different from HPASMCs and HLFs (4.1 ? 1.2 vs. 5.2 ? 1.9
[HPASMCs] and 3.6 ? 1.1 [HLFs] a.u., p = 0.75, N = 3-5/condition). Furthermore, anti-CD31,
anti-vWF, and anti-?-SMA ICC signal detection was observed in all cell types (Fig 2B),
suggesting that this methodology was invalid for confirming the identity of the isolated presumed
Determining cell type by immunofluorescence
Presumed rat PMVECs as well as HPAECs and HLFs were fixed in ice-cold acetone or
methanol and incubated for 60 min at room temperature in blocking solution (1% BSA, 10% goat
serum, and 0.1% Tween-20 in PBS). Cells were then labeled with antibodies against CD31 (Ab
#1, dilution 1:100), vWF (Ab #6, dilution 1:400), rat endothelial cell antigen-1 (RECA-1) (Ab
#8, dilution 1:100), ?-SMA (Ab #7, dilution 1:100) and vimentin (Ab #9, dilution 1:250).
Samples were incubated with a secondary antibody conjugated to Alexa Fluor 647 or 488 (Abs #10,
#11) (dilution 1:200?1:500) prior to visualization using confocal microscopy (Zeiss LSM-800,
20X Objective). Corrected total cell fluorescence was measured in a.u. using identical
microscope and laser settings for each marker in 3?8 randomly-selected fields/condition.
Colocalization was quantified in Zen Blue (Zeiss) by the Manders correlation coefficient using
singlecolor controls to determine the threshold of colocalization for each fluorophore (S2 Fig) [
Similar levels of CD31 signal were observed between presumed rat PMVECs and HPAECs
(34.8 ? 7.8 vs. 34.0 ? 3.8 a.u., p = 1.0, N = 6-8/condition). The CD31 level was significantly
higher in presumed rat PMVECs than in HPASMCs (34.8 ? 7.8 vs. 1.3 ? 0.3 a.u., p = 0.03,
N = 3-8/condition) or HLFs (34.8 ? 7.8 vs. 0.8 ? 0.3 a.u., p = 0.02, N = 3-8/condition). To
support these findings, we tested vWF as a second endothelial marker. However, we observed no
significant difference in vWF levels between presumed rat PMVECs and HPASMCs (3.8 ? 2.8
vs. 0.7 ? 0.2 a.u., p = 0.62, N = 3-4/condition) or HLFs (3.8 ? 2.8 vs. 0.2 ? 0.4 a.u., p = 0.50,
N = 3-4/condition). Although significant vWF signal was not observed in presumed rat
PMVECs relative to non-endothelial controls, vWF did appear to co-localize with CD31
compared to HPASMCs (0.71 ? 0.13 vs. 0.06 ? 0.06, Manders colocalization coefficient, p = 1.32 x
10?4, N = 6) and HLFs (0.71 ? 0.13 vs. 0.06 ? 0.06, Manders colocalization coefficient, p = 1.28
6 / 18
Fig 2. Immunocytochemistry and immunofluorescence were unsuccessful for definitively identifying presumed pulmonary microvascular endothelial cells
(PMVECs) isolated from rats ex vivo. (A) Peripheral rat lung tissue underwent mechanical and enzymatic dissociation, and the cell pellet was cultured in
endothelialselective medium. Presumed rat PMVECs were analyzed using anti-CD31 Abs #1 and #2 immunocytochemistry (ICC). Luminosity was normalized to IgG control. (B)
Anti-CD31 Ab #1, anti-von Willebrand factor (vWF) Ab #6, and anti-smooth muscle actin (?-SMA) Ab #7 ICC performed in presumed rat PMVECs, human lung
fibroblasts (HLFs) and human pulmonary artery smooth muscle cells (HPASMCs) shows no significant difference in signal intensity by cell marker across different cell
types. (C) Presumed rat PMVECs and HPAECs, HPASMCs, as well as HLFs as controls were analyzed by immunofluorescence (IF). Only CD31, and not vWF, signal
was increased in presumed rat PMVECs compared to non-endothelial controls. Representative photomicrographs shown. a.u., arbitrary units. Student?s unpaired t-test
or ANOVA. Means ? SE, N = 3-5/condition.
x 10?4, N = 6). In presumed rat PMVECs, methanol fixation was not associated with a change
in CD31-vWF co-localization relative to acetone fixation (0.72 ? 0.08 vs. 0.71 ? 0.13, Manders
colocalization coefficient, p = 0.93, N = 3) (S2 Fig).
Compared to CD31, RECA-1 expression was weak in presumed rat PMVECs. Furthermore,
inconsistent signal was observed in experimental iterations. Indeed, a statistically significant
difference in RECA-1 signal was not observed in presumed rat PMVECs compared with
HPASMCs or HLFs (11.9 ? 4.5 vs. 1.6 ? 0.2 vs. 1.1 ? 0.2, a.u., p = 0.08, N = 3/condition) (Fig
7 / 18
Cell identification by flow cytometry: Establishing a positive control
Our experiments using ICC and IF aimed to confirm the identity of cells isolated from rats ex
vivo using two standard lineage markers for ECs, but produced conflicting results. Therefore,
flow cytometry was performed next (Fig 1B). To establish a positive control for the
identification of pulmonary endothelial cells by flow cytometry, cultured HPAECs, HPASMCs, and
HLFs were grown to confluence, lifted with Accutase (Thermo Fisher, catalog #A1110501),
and blocked with 10% BSA in PBS for 15 min at 4?C. Next, cells were incubated with
directlyconjugated fluorescent antibody against CD31 (Ab #12, 3 ?g/mL), CD144 (Ab #13, 4.5 ?g/
mL), or isotype control (Ab #15, 3.0 ?g/mL; Ab #14 4.5 ?g/mL) for 45 min at 4?C, washed, and
resuspended in 250 ?L 1% BSA in PBS for immediate analysis (details on flow cytometry
methods are provided in the S1 Supporting Information).
We observed double CD31 + CD144 positivity for 80.9 ? 1.9% of HPAECs compared to
0 ? 0% of HLFs (p = 2.7 x 10?5, N = 4/condition) and 3.6 ? 1.2% of HPASMCs (p = 3.6 x 10?8,
N = 4/condition) (Fig 3A). These results were not reproduced by alternative antibodies
targeting endothelial-specific epitopes: anti-CD31 Ab #16 (18 ?g/mL) and anti-CD144 Ab #17 (9 ?g/
mL) identified marker positivity for 0.4 ? 0.1% and 30.1 ? 11.6% of HPAECs, respectively
(N = 5/Ab) (isotype controls were Ab #14 and Ab #18 for CD31 and CD144, respectively)
Fig 3. Identifying human pulmonary artery endothelial cells (HPAECs) by flow cytometry. (A) Commercially purchased HPAECs were analyzed by flow cytometry
using anti-CD31 and anti-CD144 Abs #12 and #13. Compared with HPASMCs and HLFs, high expression of CD31 and CD144 was observed only in HPAECs. These
results served as a positive control for further experiments aiming to confirm that cells isolated from rat lungs ex vivo were, in fact, endothelial. (B) Commercially
purchased HPAECs were used to test the generalizability of these results. Alternative anti-CD31 and anti-CD144 antibodies did not reliably identify endothelial cell
surface markers, supporting our earlier findings indicating variability in reactivity (i.e., quality) of tested antibodies across experimental methods, including flow
cytometry. Representative plots and histograms shown. Means ? standard error, % CD31 or CD144 positive, N = 4-5/condition. Ab, antibody; Iso, Isotype control.
8 / 18
(Fig 3B). These findings reinforced the importance of validating specific antibodies targeting
the same protein for flow cytometry to optimize results, and provided a positive control for
additional flow cytometry experiments focusing on identification of presumed rat PMVECs.
Cell identification by flow cytometry: Confirming isolated rat PMVEC
Validation of endothelial surface markers for flow cytometry. There are limited data on
the use of an optimal antibody labeling strategy for the immunophenotyping of rat lung
endothelial cells by flow cytometry. Therefore, we tested a range of endothelial surface marker
antibodies on presumed rat PMVECs. Commercially purchased RPAECs served as a positive
Presumed rat PMVECs isolated from normal rat lungs (as described in the S1 Supporting
Information) were labelled with antibodies against CD31 (Ab #12 or #16, 18 ?g/mL), CD144
(Ab #13, 18 ?g/mL) or isotype control (Ab #15, Ab #14, 18 ?g/mL), as these were the
antibodies that effectively identified HPAECs by flow cytometry. Although rat is not listed as a target
species for Ab #13 by its vendor, we nonetheless included it in this experiment because we
observed wide variability in antibody reactivity across species for many antibodies. For
example, Ab #12 is not recommended by the vendor to label human endothelial cells but did so
effectively (Fig 3A). In presumed rat PMVECs, no meaningful labeling was observed for CD31
or CD144 by Ab # 12, 16, and 13, respectively, compared to isotype control (Fig 4A). Similarly,
Ab #12 and #16 did not result in meaningful labeling of commercial RPAECs compared to
isotype control (Figs 4B and 4C). In the case of Ab #19, a high degree of false-positive, but not
true-positive, target labeling was observed despite using a conservative blocking step to
attenuate non-specific signal (10% BSA) (Fig 4D).
Ultimately, it was determined that anti-CD31 Ab #20 (R&D Biosystems, catalog #FAB3628P)
labeled commercial RPAECs effectively and selectively relative to isotype control (Ab #21, R & D
Biosystems, catalog #IC108P) (66.7 ? 14.0 vs. 0.6 ? 0.3, % CD31 positive, p = 0.003, N =
4/condition, 20 ?g/mL) or RPAMSCs (66.7 ? 14.0 vs. 1.4 ? 0.7, % CD31 positive, p = 0.003, N =
4/condition, 20 ?g/mL) (Fig 5A).
Magnetic bead-based cell isolation. Based on our findings demonstrating that anti-CD31
Ab #20 labelled commercially purchased RPAECs successfully, we next utilized this antibody
to isolate presumed normal rat PMVECs using coated magnetic beads. Importantly, this
approach is not contingent on population expansion in vitro, which, in turn, is associated with
a shift in the molecular phenotype of cells [
]. A total of 100 ?L of resuspended magnetic
bead solution (Cellection Pan Mouse IgG Kit, Thermo Fisher, catalog #11531D) was incubated
with 17 ?g/ml anti-CD31 antibody (Ab #22) and washed in 0.1% BSA in PBS per the
manufacturer directions and according to methods detailed in the S1 Supporting Information.
FACS preparation. The method for FACS preparation is outlined in the S1 Supporting
Information. Presumed rat PMVECs demonstrated strong positivity for CD31 relative to
isotype control (90.8 ? 3.7 vs. 1.4 ? 0.9, % positive for CD31, p = 1.9 x 10?5, N = 3/condition)
(Fig 5B). By contrast, no significant CD31 labeling was observed in commercial RPASMCs
(90.8 ? 3.7 vs. 0.1 ? 0.03, % positive for CD31, p = 1.6 x 10?5, N = 3/condition) and RLFs
(90.8 ? 3.7 vs. 0 ? 0, % positive for CD31, p = 0.002, N = 3/condition) (Fig 5B)
Given the challenges associated with poor antibody precision, we co-labeled cells with
GS-IB4 as a strategy to enhance the specificity of our cell yield. Specifically, GS-IB4 binds
?-Dgalactosyl residues, and has been shown previously to preferentially label endothelial cells of
microvascular origin using IF and flow cytometry [
]. Presumed rat PMVECs were
blocked as described previously and incubated with 20 ?g/mL anti-CD31 antibody #20, 5 ?g/mL
9 / 18
Fig 4. Identifying rat pulmonary endothelial cells by flow cytometry. Presumed rat PMVECs isolated by mechanical and enzymatic dissociation of peripheral lung
and culture in endothelial-selective medium, commercial rat pulmonary artery endothelial cells (RPAECs), or rat pulmonary artery smooth muscle cells (RPASMCs)
were labeled with antibodies against endothelial surface markers. (A) Anti-CD31 (Ab #12 and #16) and CD144 (Ab #13) signal was not observed in presumed rat
PMVECs by flow cytometry (N = 3/condition). (B) Labeling of RPAECs was also not observed for anti-CD31 antibodies #12 (N = 4/condition) and (C) #16, respectively
(N = 3/condition). (D) False-positive signal was detected in RPASMCs labeled with anti-CD144 Ab #19 (N = 4/condition). Representative plots and histograms shown.
Means ? standard error, % CD31 or CD144 positive. Ab, antibody; Iso, Isotype control.
GS IB4, or control (antibodies #21 or #15, respectively) for 45 min at 4?C in a total of volume
of 50 ?L. Compared to IgG control Ab #14, GS-IB4 signal was strongly positive in presumed
rat PMVECs (0.5 ? 0.2 vs. 77.1 ? 8.8, % GS IB4 positive, p = 9.3 x 10?4, N = 3/condition).
Compared to presumed rat PMVECs, we observed no meaningful GS-IB4 labeling in
commercial RPASMCs (77.1 ? 8.8 vs. 0.1 ? 0.1, % GS-IB4 positive, p = 9.2 x 10?4, N = 3/condition,
5 ?g/mL) and RLFs (77.1 ? 8.8 vs. 2.1 ? 0.6, % GS-IB4 positive, p = 0.001, N = 3/condition,
5 ?g/mL) (Fig 5C). Furthermore, 93.8 ? 2.8% of CD31-positive cells also labeled positively for
GS-IB4 (5 ?g/mL, N = 3/condition) (Fig 5D). By propidium iodide analysis, GS-IB4 labeling
did not adversely influence the viability of presumed rat PMVECs (86.6 ? 7.7 vs. 86.9 ? 6.9, %
viable cells, p = 0.98, N = 3/condition).
Preparing rat PMVECs isolated by FACS for transcriptomic analyses. Overall,
CD31-positive cells comprised 86.7 ? 2.9% (N = 3) of the total cell population isolated from rat
lungs by magnetic bead selection. Although this is consistent with published data on other
endothelial cell types, it is possible that including cells that were not positive for CD31 could
adversely influence results of subsequent transcriptomic analyses [
]. Thus, we confirmed rat
PMVEC identity by virtue of CD31 + GS-IB4 double-positivity sorted by FACS. Only that cell
population was used for isolating RNA in preparation for transcriptomic analyses. The
FACSAria Special Order flow cytometer was used for cell sorting, as detailed in the S1 Supporting
10 / 18
Fig 5. Fluorescence-activated cell sorting (FACS) permits isolation of rat pulmonary microvascular endothelial cells (PMVECs). Presumed rat PMVECs isolated
from peripheral lung by immunomagnetic anti-CD31 bead selection, as well as commercial rat pulmonary artery endothelial cells (RPAECs), rat lung fibroblasts (RLFs)
and RPASMCs were analyzed by flow cytometry directed against endothelial surface markers. (A) Anti-CD31 Ab #20 selectively labeled RPAECs relative to isotype
control and RPASMCs (N = 4/condition). (B) Presumed rat PMVECs demonstrate specific anti-CD31 labeling relative to commercial rat lung fibroblasts (RLFs) and
RPASMCs (N = 3/condition). (C) Presumed rat PMVECs also demonstrate specific signal for isolectin 1-B4 from Griffonia simplicifolia (GS-IB4) relative to RLFs and
RPASMCs (N = 4/condition). (D) Over 90% of CD31-positive presumed rat PMVECs co-label with GS-IB4 (N = 3/condition). Confirmed rat PMVECs were defined as
those cells positive for both CD31 and GS-IB4 by flow cytometry. Representative plots and histograms shown. Means, % CD31 or GS-IB4 positive. Ab, antibody; Iso,
To demonstrate that immunomagnetic bead selection for CD31 followed by FACS isolation
of CD31 + GS-IB4 double-positive cells is a valid method of isolating high-quality RNA for
transcriptomic profiling in experimental PAH, male Sprague Dawley rats were administered a single
intraperitoneal injection of monocrotaline (MCT) (Sigma C2401-1G) or normal saline control
on day 0 of the protocol. Confirmed rat PMVECs comprised 78.1 ? 5.8% and 61 ? 7.3% of viable
presumed PMVECs, in control and MCT-PAH, respectively (p = 0.1) (S3 Fig). RNA was isolated
from confirmed MCT-PAH PMVECs on day 23 and assayed for quality (Agilent 2100
Bioanalyzer). RNA integrity number (RIN) (Agilent) was 9.2 ? 0.1 vs. 8.7 ? 0.1 (p = 0.003) for control vs.
11 / 18
MCT-PAH, respectively (N = 6 rats/condition) (Table 2). Full RNA electropherograms are
available for all samples in S2 Supporting Information.
In this report, we detail a novel approach to isolating PMVECs directly from rat lungs using
flow cytometry. Specifically, magnetic bead selection for CD31 followed by FACS identified a
high rate of double positive cells for CD31 and GS-IB4, which enhanced the specificity of cell
recovery and permitted isolation of high-quality RNA from PMVECs without culturing cells.
This allows for next generation transcriptomic analyses on PMVECs ex vivo without passaging,
which is associated with changes in genomic and proteomic programing that may limit
translational relevance of results [
]. Our data also illustrate discrepancies in the quality of
commercially available and commonly referenced biomaterials that are used to identify cell type.
Thus, a second major finding of this work relates to the critical importance of
investigator-initiated validation of reagents used in PAH and other experimental biology fields.
Studying PAECs isolated from patients is ideal for experimental research, but PAH is a rare
disease, limiting donor availability from explanted lungs, and accessing distal pulmonary
arterials using minimally invasive methods is associated with risk. Isolating and phenotyping
primary human or murine pulmonary endothelial cells has been reported previously using serial
passaging with endothelial cell-selective media, anti-endothelial antibody-coated magnetic
beads, or FACS [
]. However, experimental murine models do not
recapitulate many features of PAH observed in patients, particularly plexogenic vascular lesions or
severe pulmonary hypertension. By contrast, there are few reports focusing on methods for
isolating pulmonary artery endothelial cells in rats despite important advantages of PAH
models in this species. This may partly reflect inconsistent quality for rat-compatible commercially
available anti-endothelial antibodies. For example, the anti-CD31 monoclonal antibody clone
TLD-3A12 is commonly referenced as a rat lung endothelial marker [
]. But in our
experience, this clone was ineffective for profiling presumed PMVECs by ICC or flow
Our findings show that Ab #12, which is also derived from clone TLD-3A12, had excellent
sensitivity and specificity for the detection of CD31 on HPAECs by flow cytometry.
Importantly, Homo sapiens is not listed as a target species by the antibody vendor. This observation is
consistent with limited access to (or availability of) data on the target antigen or derivative
epitope for that clone specifically, as well as other antibodies more generally, which ultimately
confounds predicting relevant biophysical interactions that may explain experimental results.
Other antibodies against endothelial surface antigens were associated with poor specificity.
For example, in some experiments we observed strong CD31 and vWF expression in
Confirmed PMVEC RNA was isolated from monocrotaline (MCT) and control-treated rats, and the RNA Integrity
Number (RIN) was determined on an Agilent 2100 Bioanalyzer. PAH, pulmonary arterial hypertension.
12 / 18
HPASMCs. This trend was not limited to ICC or IF analytical methods: a false detection rate
for CD144 positive cells was observed in >20% RPASMCs analyzed by flow cytometry, which
is consistent with published reports from others [
Taken together, these data suggest that the recognition of pulmonary endothelial antigens by
commercially available antibodies is variable and assay-dependent. This experience is in concert
with accumulating data implicating reagent quality in the synthesis of low fidelity or
irreproducible findings [
]. Our data expand this field by cataloguing the pervasiveness and extent of this
problem across a wide spectrum of methodologies and antibodies that are purported to share
the same target. Although these findings clarify the importance of using valid reagents for
fundamental experimentation (i.e., cell type identification), a simple solution to this problem
appears less certain. Antibody validation imposes a substantial financial and time burden on
scientific investigators. For example, over the course of this project, antibody validation
experiments consumed an estimated $69,096 in reagent, personnel, and FACS expenses?$9,545
(13.8%) of which was spent on the purchase of commercial antibodies (Fig 6). Reagent
validation may, thus, warrant greater consideration when considering laboratory budgets by
investigator and sponsor alike.
The intent of this project was to isolate high-quality mRNA from rat PMVECs successfully,
and, therefore, an exhaustive evaluation of all commercially available products and labeling
conditions was not performed. For example, antibodies against intracellular antigens were not
tested since this necessitates cell fixation and/or permeabilization for FACS. However, others
have reported that efficient RNA recovery from fixed cells may be feasible [
most cells and biomaterials were fresh, our experiments did not control for age and variability
in storage conditions, which may have affected our results. We recognize that antibody labeling
experiments typically require optimization; it is possible that different extracellular matrix
coatings in vitro, cell culture conditions, and methods of fixation or immunolabeling could
influence the results of the IHC, IF, and flow cytometry analyses. Significant RECA-1 expression was
not observed by IF in presumed rat PMVECs. The mechanism of this observation was not
explored, as we ultimately favored a FACS-based rat PMVEC isolation strategy that was not
contingent on RECA-1 labeling. In general, we followed vendor-recommended antibody
labeling protocols, and performed additional attempts at optimization when possible. Budgetary and
time constraints precluded an independent evaluation of the purity of each lot of commercial
] although each primary cell type was analyzed by flow cytometry.
Further experiments are needed to confirm that our methodology isolated PAECs
exclusively. Others have reported CD31 and ?-D-galactosyl surface residue presence in
non-endothelial cells, and distinguishing endothelial cells of vascular versus lymphatic origin was not a
specific focus of this study [
]. However, it has been shown previously that GS-IB4
does not co-localize with lymphatic endothelial-specific cells (defined by LYVE-1 expression)
in the lung, providing indirect evidence to cell endothelial populations isolated in our report
are predominately vascular in origin [
]. Additionally, we used flow cytometry gating
settings that were conservative, and focused on populations with the highest CD31 and GS-IB4
signals to minimize the chance for detection of other cell types [
]. Nonetheless, alternative
proteins, such as endoglin and vascular endothelial growth factor receptor-2, may provide
enhanced endothelial specificity and should be considered in future research.
In summary, we present a methodology for isolating PMVECs from rats ex vivo using flow
cytometry that does not require cell culture or passaging prior to transcriptomic analysis. We
identified poor sensitivity and specificity of commercially available antibodies for pulmonary
endothelial antigens. These collective findings have important implications for future work in
experimental PAH, particularly translational endeavors that involve interrogating the ?omic?
13 / 18
Fig 6. Reagent, personnel, and equipment costs attributable to the validation of commercial biomaterials. Distribution of expenses attributable to the validation of
commercial products used in the isolation of rat pulmonary microvascular endothelial cells. FACS, fluorescence-activated cell sorting.
profile for PMVECs, and underscores the need for investigator-driven validation of key
S1 Supporting Information. This document contains details regarding flow cytometry
setup, endothelial cell isolation methods, as well as sources and characteristics of
commercial primary cells.
S2 Supporting Information. Raw electropherograms for rat PMVEC RNA isolated from
control and monocrotaline-PAH animals are provided in this document.
S1 Fig. Detection of CD31 and ?-smooth muscle actin by immunocytochemistry was
similar with acetone or methanol cell fixation. Peripheral rat lung tissue was treated with
mechanical and enzymatic dissociation, and the cell pellet was cultured in endothelial-selective
medium. Presumed rat PMVECs were fixed in acetone or methanol and analyzed using
antiCD31 Ab #1 and anti-?-smooth muscle actin Ab # 7 immunocytochemistry. Luminosity was
normalized to IgG (Ab #3). Representative images shown. a.u., arbitrary units. Student?s
14 / 18
unpaired t-test. Means ? SE, N = 3/condition.
S2 Fig. CD31 and von Willebrand Factor colocalize in human pulmonary arterial
endothelial cells and presumed rat pulmonary microvascular endothelial cells. (A) Human
pulmonary artery endothelial cells were labeled with either anti-CD31 Ab #1 or anti-von Willebrand
Factor Ab #6 and analyzed using confocal microscopy to determine colocalization thresholds.
(B) Peripheral rat lung tissue was subjected to mechanical and enzymatic dissociation, and the
cell pellet was cultured in endothelial-selective medium. Presumed rat PMVECs, human
pulmonary artery endothelial cells, human pulmonary artery smooth muscle cells, and human
lung fibroblasts were fixed in acetone and co-labeled with anti-CD31 Ab #1 and anti-von
Willebrand Factor Ab #6 and colocalization was measured using the thresholds established in
panel (A). To enhance visualization, regions of colocalization are emphasized using a
false-colored yellow overlay. (C) Meaningful differences in CD31-vWF colocalization were not
observed between methanol and acetone fixation of presumed rat PMVECs. Representative
images and scatterplots shown. AF 488, Alexa Fluor 488; AF 647, Alexa Fluor 647. Student?s
unpaired t-test. Means ? SE, N = 3/condition.
S3 Fig. Detailed gating strategy for the identification of confirmed rat pulmonary
microvascular endothelial cells by CD31 and Griffonia simplicifolia isolectin 1-B4 flow
cytometry. Presumed rat PMVECs were isolated without cell culture by mechanical and enzymatic
digestion and immunomagnetic bead selection for CD31. Presumed rat PMVECs were labeled
with anti-CD31 Ab #20 (conjugated to phycoerythrin) and Griffonia simplicifolia isolectin
1-B4 (conjugated to Alexa Fluor 488) and analyzed by flow cytometry. Fluorescence minus one
controls were used to establish gates. Isotype or IgG control confirmed the specificity of cell
labeling by Griffonia simplicifolia isolectin 1-B4. Viability was assessed by propidium iodide.
Representative plots shown. AF 488, Alexa Fluor 488; AF 647, Alexa Fluor 647; FSC-H,
forward scatter-height; PE, phycoerythrin; PI, propidium iodide; SSc-A, side scatter-area.
The authors acknowledge Teresa Dinter and Brad Dykstra for technical assistance regarding
rat endothelial cell isolation and flow cytometry, respectively. The authors also acknowledge
Ronglih Liao for her review of the manuscript, as well as the services of the Brigham and
Women?s Hospital Flow Cytometry Core Laboratory.
Conceptualization: Bradley M. Wertheim, Yi-Dong Lin, Andriy O. Samokhin, Paul B. Yu,
Bradley A. Maron.
Data curation: Bradley M. Wertheim, Yi-Dong Lin, Bradley A. Maron.
Formal analysis: Bradley M. Wertheim, Yi-Dong Lin, Bradley A. Maron.
Funding acquisition: Bradley M. Wertheim, Paul B. Yu, Bradley A. Maron.
Investigation: Bradley M. Wertheim, Yi-Dong Lin, Ying-Yi Zhang, Andriy O. Samokhin,
George A. Alba, Paul B. Yu, Bradley A. Maron.
Methodology: Bradley M. Wertheim, Yi-Dong Lin, Ying-Yi Zhang, Andriy O. Samokhin,
George A. Alba, Elena Arons, Paul B. Yu, Bradley A. Maron.
15 / 18
Project administration: Bradley M. Wertheim, Elena Arons, Bradley A. Maron.
Resources: Bradley M. Wertheim, Paul B. Yu, Bradley A. Maron.
Software: Bradley M. Wertheim, Yi-Dong Lin, Bradley A. Maron.
Supervision: Bradley A. Maron.
Validation: Bradley M. Wertheim, Yi-Dong Lin, Bradley A. Maron.
Visualization: Bradley M. Wertheim, Yi-Dong Lin, Bradley A. Maron.
Writing ? original draft: Bradley M. Wertheim, Bradley A. Maron.
Writing ? review & editing: Bradley M. Wertheim, Yi-Dong Lin, Ying-Yi Zhang, Andriy O.
Samokhin, George A. Alba, Elena Arons, Paul B. Yu, Bradley A. Maron.
16 / 18
17 / 18
1. Samokhin AO , Stephens T , Wertheim BM , Wang R-S , Vargas SO , Yung L-M , et al. NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension . Sci Transl Med . 2018 ; 10 . https://doi.org/10.1126/scitranslmed.aap7294 PMID: 29899023
2. Pollett JB , Benza RL , Murali S , Shields KJ , Passineau MJ . Harvest of pulmonary artery endothelial cells from patients undergoing right heart catheterization . J Heart Lung Transplant . 2013 ; 32 : 746 - 749 . https://doi.org/10.1016/j.healun. 2013 . 04 .013 PMID: 23684132
3. Benza RL , Williams G , Wu C , Shields KJ , Raina A , Murali S , et al. In Situ Expression of Bcl-2 in Pulmonary Artery Endothelial Cells Associates with Pulmonary Arterial Hypertension Relative to Heart Failure with Preserved Ejection Fraction . Pulm Circ . 2016 ; 6 : 551 - 556 . https://doi.org/10.1086/688774 PMID: 28090298
4. Provencher S , Archer SL , Ramirez FD , Hibbert B , Paulin R , Boucherat O , et al. Standards and Methodological Rigor in Pulmonary Arterial Hypertension Preclinical and Translational Research. Circ Res . 2018 ; 122 : 1021 - 1032 . https://doi.org/10.1161/CIRCRESAHA.117.312579 PMID: 29599278
5. Suresh K , Servinsky L , Jiang H , Bigham Z , Yun X , Kliment C , et al. Reactive oxygen species induced Ca2+ influx via TRPV4 and microvascular endothelial dysfunction in the SU5416/hypoxia model of pulmonary arterial hypertension . Am J Physiol Lung Cell Mol Physiol . 2018 ; 314 : L893 - L907 . https://doi. org/10.1152/ajplung.00430. 2017 PMID: 29388466
6. Comhair SAA , Xu W , Mavrakis L , Aldred MA , Asosingh K , Erzurum SC . Human Primary Lung Endothelial Cells in Culture . Am J Respir Cell Mol Biol . 2012 ; 46 : 723 - 730 . https://doi.org/10.1165/rcmb.2011 - 0416TE PMID: 22427538
7. King J , Hamil T , Creighton J , Wu S , Bhat P , McDonald F , et al. Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes . Microvasc Res . 2004 ; 67 : 139 - 151 . https://doi.org/10.1016/j.mvr. 2003 . 11 .006 PMID: 15020205
8. Sahagun G , Moore SA , Fabry Z , Schelper RL , Hart MN . Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled griffonia simplicifolia agglutinin . Am J Pathol . 1989 ; 134 : 1227 - 1232 . PMID: 2757116
9. Gaskill C , Majka SM. A high-yield isolation and enrichment strategy for human lung microvascular endothelial cells . Pulm Circ . 2017 ; 7 : 108 - 116 . https://doi.org/10.1177/2045893217702346 PMID: 28680570
10. Alphonse RS , Vadivel A , Zong S , McConaghy S , Ohls R , Yoder MC , et al. The isolation and culture of endothelial colony-forming cells from human and rat lungs . Nat Protoc . 2015 ; 10 : 1697 - 1708 . https:// doi.org/10.1038/nprot. 2015 .107 PMID: 26448359
11. Jin Y , Liu Y , Antonyak M , Peng X . Isolation and Characterization of Vascular Endothelial Cells from Murine Heart and Lung . In: Peng X , Antonyak M , editors. Cardiovascular Development . Totowa, NJ: Humana Press; 2012 . pp. 147 - 154 . https://doi.org/10.1007/978-1- 61779 -523-7_ 14
12. Sobczak M , Dargatz J , Chrzanowska-Wodnicka M . Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice . J Vis Exp . 2010 ; https://doi.org/10.3791/2316 PMID: 21178973
13. Weber SC , Gratopp A , Akanbi S , Rheinlaender C , Sallmon H , Barikbin P , et al. Isolation and culture of fibroblasts, vascular smooth muscle, and endothelial cells from the fetal rat ductus arteriosus . Pediatr Res . 2011 ; 70 : 236 . https://doi.org/10.1203/PDR.0b013e318225f748 PMID: 21629157
14. Lu H , Yuan H , Chen S , Huang L , Xiang H , Yang G , et al. Senescent endothelial dysfunction is attributed to the up-regulation of sphingosine-1-phosphate receptor-2 in aged rats . Mol Cell Biochem . 2012 ; 363 : 217 - 224 . https://doi.org/10.1007/s11010-011-1173-y PMID: 22139303
15. Fehrenbach ML , Cao G , Williams JT , Finklestein JM , DeLisser HM . Isolation of murine lung endothelial cells . Am J Physiol-Lung Cell Mol Physiol . 2009 ; 296 : L1096 - L1103 . https://doi.org/10.1152/ajplung. 90613. 2008 PMID: 19304908
16. van Beijnum JR , Rousch M , Castermans K , van der Linden E , Griffioen AW . Isolation of endothelial cells from fresh tissues . Nat Protoc . 2008 ; 3 : 1085 - 1091 . https://doi.org/10.1038/nprot. 2008 .71 PMID: 18546599
17. Magee JC , Stone AE , Oldham KT , Guice KS . Isolation, culture, and characterization of rat lung microvascular endothelial cells . Am J Physiol-Lung Cell Mol Physiol . 1994 ; 267 : L433 - L441 .
18. Ryan US , White LA , Lopez M , Ryan JW . Use of microcarriers to isolate and culture pulmonary microvascular endothelium . Tissue Cell . 1982 ; 14 : 597 - 606 . PMID: 6815827
19. Kim J , Kang Y , Kojima Y , Lighthouse JK , Hu X , Aldred MA , et al. An endothelial apelin-FGF link mediated by miR-424 and miR-503 is disrupted in pulmonary arterial hypertension . Nat Med . 2013 ; 19 : 74 - 82 . https://doi.org/10.1038/nm.3040 PMID: 23263626
20. Mitchell JB , McIntosh K , Zvonic S , Garrett S , Floyd ZE , Kloster A , et al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers . Stem Cells Dayt Ohio . 2006 ; 24 : 376 - 385 . https://doi.org/10.1634/stemcells.2005-0234 PMID: 16322640
21. Collins FS , Tabak LA . NIH plans to enhance reproducibility . Nature . 2014 ; 505 : 612 . PMID: 24482835
22. Lauer M. Authentication of Key Biological and/or Chemical Resources in NIH Grant Applications . In: NIH Extramural Nexus [Internet]. 29 Jan 2016 [cited 8 Jun 2018 ]. Available: https://nexus.od.nih.gov/all/ 2016/01/29/authentication-of -key-biological-andor-chemical-resources-in-nih-grant-applications/
23. Bradbury A , Pluckthun A . Standardize antibodies used in research: to save millions of dollars and dramatically improve reproducibility, protein-binding reagents must be defined by their sequences and produced as recombinant proteins, say Andrew Bradbury, Andreas Pluckthun and 110 co-signatories . Nature . 2015 ; 518 : 27 - 30 . https://doi.org/10.1038/518027a PMID: 25652980
24. Kosmidou C , Efstathiou NE , Hoang MV , Notomi S , Konstantinou EK , Hirano M , et al. Issues with the Specificity of Immunological Reagents for NLRP3: Implications for Age-related Macular Degeneration . Sci Rep . 2018 ; 8 . https://doi.org/10.1038/s41598-017 -17634-1 PMID: 29323137
25. Berglund L , Bjo?rling E, Oksvold P , Fagerberg L , Asplund A , Szigyarto CA-K , et al. A genecentric Human Protein Atlas for expression profiles based on antibodies . Mol Cell Proteomics . 2008 ; 7: 2019 - 2027 . https://doi.org/10.1074/mcp.R800013 -MCP200 PMID : 18669619
26. Voskuil JLA . The challenges with the validation of research antibodies . F1000Research . 2017 ; 6 : 161 . https://doi.org/10.12688/f1000research.10851.1 PMID: 28357047
27. Ranchoux B , Harvey LD , Ayon RJ , Babicheva A , Bonnet S , Chan SY , et al. Endothelial dysfunction in pulmonary arterial hypertension: an evolving landscape (2017 Grover Conference Series) . Pulm Circ . 2018 ; 8 : 2045893217752912 . https://doi.org/10.1177/2045893217752912 PMID: 29283043
28. Szulcek R , Happe? CM, Rol N , Fontijn RD , Dickhoff C , Hartemink KJ , et al. Delayed Microvascular Shear Adaptation in Pulmonary Arterial Hypertension. Role of Platelet Endothelial Cell Adhesion Molecule-1 Cleavage. Am J Respir Crit Care Med . 2016 ; 193 : 1410 - 1420 . https://doi.org/10.1164/rccm. 201506-1231OC PMID: 26760925
29. Zinchuk V , Zinchuk O , Okada T. Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena . ACTA Histochem Cytochem . 2007 ; 40 : 101 - 111 . https://doi.org/10.1267/ahc.07002 PMID: 17898874
30. Townsley MI . Structure and Composition of Pulmonary Arteries, Capillaries, and Veins . In: Terjung R, editor. Comprehensive Physiology . Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2012 . https://doi.org/ 10.1002/cphy.c100081
31. Hansen-Smith FM , Watson L , Lu DY , Goldstein I. Griffonia simplicifolia I: fluorescent tracer for microcirculatory vessels in nonperfused thin muscles and sectioned muscle . Microvasc Res . 1988 ; 36 : 199 - 215 . PMID: 3148100
32. Helenius MH , Vattulainen S , Orcholski M , Aho J , Komulainen A , Taimen P , et al. Suppression of endothelial CD39/ENTPD1 is associated with pulmonary vascular remodeling in pulmonary arterial hypertension . Am J Physiol-Lung Cell Mol Physiol . 2015 ; 308 : L1046 - L1057 . https://doi.org/10.1152/ajplung. 00340. 2014 PMID: 25820525
33. Tamosiuniene R , Tian W , Dhillon G , Wang L , Sung YK , Gera L , et al. Regulatory T Cells Limit Vascular Endothelial Injury and Prevent Pulmonary Hypertension. Circ Res . 2011 ; 109 : 867 - 879 . https://doi.org/ 10.1161/CIRCRESAHA.110.236927 PMID: 21868697
34. Liu L , Nielsen FM , Riis SE , Emmersen J , Fink T , Hjortdal J? , et al. Maintaining RNA Integrity for Transcriptomic Profiling of Ex Vivo Cultured Limbal Epithelial Stem Cells after Fluorescence-Activated Cell Sorting (FACS) . Biol Proced Online . 2017 ; 19 . https://doi.org/10.1186/s12575-017 -0065-2 PMID: 29255379
35. Esser C , Go?ttlinger C , Kremer J , Hundeiker C , Radbruch A. Isolation of full-size mRNA from ethanolfixed cells after cellular immunofluorescence staining and fluorescence-activated cell sorting (FACS) . Cytometry . 1995 ; 21 : 382 - 386 . https://doi.org/10.1002/cyto.990210411 PMID: 8608737
36. Nilsson H , Krawczyk KM , Johansson ME . High salt buffer improves integrity of RNA after fluorescenceactivated cell sorting of intracellular labeled cells . J Biotechnol . 2014 ; 192 : 62 - 65 . https://doi.org/10. 1016/j.jbiotec. 2014 . 09 .016 PMID: 25277986
37. Acharya P , Quinlan A , Neumeister V . The ABCs of finding a good antibody: How to find a good antibody, validate it, and publish meaningful data . F1000Research . 2017 ; 6 : 851 . https://doi.org/10.12688/ f1000research.11774.1 PMID: 28713558
38. Watt SM , Williamson J , Genevier H , Fawcett J , Simmons DL , Hatzfeld A , et al. The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes . Blood . 1993 ; 82 : 2649 - 2663 . PMID: 7693043
39. Maddox DE , Shibata S , Goldstein IJ . Stimulated macrophages express a new glycoprotein receptor reactive with Griffonia simplicifolia I-B4 isolectin . Proc Natl Acad Sci . 1982 ; 79 : 166 - 170 . https://doi.org/ 10.1073/pnas.79.1.166 PMID: 6798567
40. Silverman JD , Kruger L . Selective neuronal glycoconjugate expression in sensory and autonomic ganglia: relation of lectin reactivity to peptide and enzyme markers . J Neurocytol . 1990 ; 19 : 789 - 801 . PMID: 2077115
41. Baluk P , McDonald DM . Markers for Microscopic Imaging of Lymphangiogenesis and Angiogenesis . Ann N Y Acad Sci . 2008 ; 1131 : 1 - 12 . https://doi.org/10.1196/annals.1413.001 PMID: 18519955
42. Moldobaeva A , Jenkins J , Zhong Q , Wagner EM . Lymphangiogenesis in rat asthma model . Angiogenesis . 2017 ; 20 : 73 - 84 . https://doi.org/10.1007/s10456-016 -9529-2 PMID: 27787629