Selecting the right gate to identify relevant cells for your assay: a study of thioglycollate-elicited peritoneal exudate cells in mice
Hermida et al. BMC Res Notes
Selecting the right gate to identify relevant cells for your assay: a study of thioglycollate-elicited peritoneal exudate cells in mice
Micely D. R. Hermida 0
Rafaela Malta 0
Marcos D. P. C. de S. Santos 0
Washington L. C. dos‑Santos 0
0 Laboratório de Patologia e Biointervenção, Fundação Oswaldo Cruz, FIOCRUZ‐ BA, Instituto Gonçalo Moniz, Rua Waldemar Falcão , no 121, Candeal, Salvador, Bahia CEP 40296‐710 , Brazil
Objective: In this study, we investigate the diversity and modulation of leukocyte populations represented in the gates defined by size and granularity at different time points of thioglycollate‑ induced peritonitis in mouse. Results: The inflammatory cells were distributed into four regions (R1-R4) of a data plot graph defined by cell size and granularity. R1 and R2 contained agranular cells that were small in size and predominately included T (CD3+) lymphocytes along with B (B220+) lymphocytes. Macrophages (F4/80+) were the predominant cells found in the R3 region. However, these cells were present in all regions, albeit at a lower frequency in R1 and R2. Granulocytes (Gr1+) were mainly distributed in R3 and R4. The wide distribution of F4/80+ and Gr1+ cells may reflect the recruitment and activation state of the different macrophage and granulocyte populations. Based on these observations, size and granularity may contribute to an initial step in the analysis and sorting of thioglycollate‑ elicited peritoneal exudate cells. However, the developmental stage and cell activation state may interfere with cell segregation using size and granularity as parameters.
Flow cytometry; Peritoneal exudate cells; Thioglycollate stimuli; Cell size and granularity
Introduction
The experimental induction of peritonitis in mice with
thioglycollate (TGM) allows a variety of leukocytes to be
obtained in large numbers under sterile conditions that
are suitable for in vitro cultivation and a variety of
experiments [
1–3
]. For example, neutrophils, macrophages and
lymphocytes may predominate during different stages of
TGM-induced peritonitis [
3, 4
]. In many studies, cell size
and granularity alone or in combination with antibody
labeling are used for the analysis and sorting of relevant
leukocyte populations by flow cytometry. The
distribution of TGM-elicited PECs when plotted by cell size and
granularity results in the visualization of at least four
distinct regions. Although these regions predominately
correspond with lymphocytes, macrophages and
polymorfonuclear leukocytes, the cell composition is diverse
and further enhanced by peritonitis progression [4].
In this work, we use morphology together with
immunophenotyping to characterize the TGM-elicited PECs
distributed in the most representative clusters defined
by size and granularity on flow-cytometric dot plots. The
aim of this work is to minimize misinterpretation of cell
analysis data by providing a strategy that takes advantage
of the cell diversity during the course of peritonitis.
Main text
Methods
Kinetics of inflammatory cell influx into the peritoneal cavity
in thioglycollate‑induced peritonitis
Peritonitis was induced in BALB/c mice, 6- to
8-weekold of both sex, by injecting 3 ml of a sterile 3% (wt/vol)
thioglycollate (catalog # T9032, Sigma Aldrich, USA)
solution. PECs were collected after 4, 8 and 12 h and after
1, 2, 4, 10, 20, 40 and 100 days by washing the peritoneal
cavity twice with cold Ca2+ and Mg2+-free Hanks’
balanced salt solution (HBSS; Sigma Aldrich, USA)
containing 20 IU/ml heparin. The number of cells collected from
each animal was estimated using a Neubauer chamber.
Cell viability was assessed by trypan blue dye exclusion,
and cell populations were defined by morphology using
cytospin preparations and specific antibodies for
detection by flow cytometry. All the experiments were
independently repeated twice using three animals in each
group.
Flow cytometry analysis
The cells (1–2 × 106/stain) were stained with the
following fluorescein isothiocyanate-conjugated
antibodies: anti-CD3e (145-2C11, catalog# 553061), anti-B220
(RA3-6B2, catalog# 553087) and anti-GR-1 (RB6-8C5,
catalog# 553126) (BD-Bioscience; USA) and
phycoerythrin (PE)-conjugated antibody was anti-F4/80
antigen (BM8.1, catalog# FP20066010; Caltag, USA) as
previously described [
1
]. Unlabeled or isotype-matched
stained cells were used as controls. The cells were
analyzed using a FACSAria III flow cytometer and FlowJo
Software (Tree Star, USA).
Cell sorter and leukocyte morphology
PECs were sorted based on their size and granularity using
a FACSAria III sorter (BD Biosciences, USA),
cytocentrifuged at 500 rpm onto glass slides and stained with
Papanicolaou’s, hematoxylin and eosin, or the Luna’s (to detect
eosinophil granules) stain techniques [
5–7
]. Leukocyte
were identified using previously defined characteristics [
8
].
Results
Kinetics of leukocyte recruitment in thioglycollate‑induced
inflammation
The (...truncated)