Selecting the right gate to identify relevant cells for your assay: a study of thioglycollate-elicited peritoneal exudate cells in mice

BMC Research Notes, Dec 2017

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.

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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)


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Micely D. R. Hermida, Rafaela Malta, Marcos D. P. C. de S. Santos, Washington L. C. dos-Santos. Selecting the right gate to identify relevant cells for your assay: a study of thioglycollate-elicited peritoneal exudate cells in mice, BMC Research Notes, 2017, pp. 695, Volume 10, Issue 1, DOI: 10.1186/s13104-017-3019-5