For immunophenotyping analysis of hematopoietic cells, spleen, thymus and bone marrow cells were isolated, RBC lysed (Pharmlyse, BD Biosciences) and analyzed for surface staining with αCD3-FITC (Clone 17A2) [44] (link), αB220-eFluor450 (Clone RA3-6B2, eBioscience), αCD4-PerCP (Clone RM4-5, Biolegend) and αCD8β-Cy5 (Clone RmCD8-2) [44] (link). Samples were analyzed using an LSRII (BD Biosciences).
For SEPT7, acetyl tubulin and propidium iodide staining, thymocytes/splenocytes were fixed with 3× by volume PFA (4%) at RT for 30 min. Washed and resuspended in PBS and absolute methanol was added to 90% concentration final with constant mixing. The methanol permeabilization was continued for 30 min on ice. After 2× PBS wash cells were resuspended in 4% BSA-PBS and blocked at 4°C for 30 min. Cells were stained with primary antibodies (1∶100 in 1%BSA-PBS) at RT for 30 min. After 1× PBS wash, samples were resuspended in secondary antibody dilution (anti rabbit Alexa fluor-488/anti mouse Alexa fluor-546 - 1∶500 diluted in 1% BSA-PBS) and incubated for additional 30 min before PBS wash and FACS analysis. For analysis of DNA content fixed cells were treated with nuclear stain solution (1× PBS, 100 µg/mL propidium iodide, 100 µg/mL RNAse-A) at RT for 15 min and analyzed by flow cytometry in Accuri- C6 flow cytometer.
For SEPT7, acetyl tubulin and propidium iodide staining, thymocytes/splenocytes were fixed with 3× by volume PFA (4%) at RT for 30 min. Washed and resuspended in PBS and absolute methanol was added to 90% concentration final with constant mixing. The methanol permeabilization was continued for 30 min on ice. After 2× PBS wash cells were resuspended in 4% BSA-PBS and blocked at 4°C for 30 min. Cells were stained with primary antibodies (1∶100 in 1%BSA-PBS) at RT for 30 min. After 1× PBS wash, samples were resuspended in secondary antibody dilution (anti rabbit Alexa fluor-488/anti mouse Alexa fluor-546 - 1∶500 diluted in 1% BSA-PBS) and incubated for additional 30 min before PBS wash and FACS analysis. For analysis of DNA content fixed cells were treated with nuclear stain solution (1× PBS, 100 µg/mL propidium iodide, 100 µg/mL RNAse-A) at RT for 15 min and analyzed by flow cytometry in Accuri- C6 flow cytometer.