Results were reported as absolute numbers of T cells per organs after performing an initial organ cell count using trypan blue and staining and acquiring a fixed number of cells. One million splenocytes or lung cells were stained using antibodies to CD3 (564380), CD8 (551162), CD4 (561025), CD62L (553150), CD44 (562464), CCR7 (12-1971-80), CD103 (562772), CD69 (740220), CD11a (740849), Live/Dead Fixable Aqua Dead Cell Stain Kit (L34957). All the antibodies were purchased by BD Biosciences. CCR7 antibody was purchased from eBiosciences and Live/Dead staining kit was purchased from Thermo Fisher Scientific. After fixation and storage at 4°C overnight, the cells were analyzed using a BD Fortessa flow cytometer. The FlowJo software program was used to analyze the data (Tree Star Inc., Stanford, CA).
For intracellular staining, cells were fixed and permeabilized according to manufacture instruction of using Fixation/Permeabilization Solution Kit with BD GolgiStop (554715: BD Biosciences) and stained for IFN-γ (564336: BD Biosciences) and IL-2 (560547: BD Biosciences). All staining antibodies were used at 1:200 or 1:300 or 1:500 dilution depending on the experiment. Sample acquisition was performed on a BD Fortessa flow cytometer using FACS Diva software followed by data analysis with FlowJo software.
For intracellular staining, cells were fixed and permeabilized according to manufacture instruction of using Fixation/Permeabilization Solution Kit with BD GolgiStop (554715: BD Biosciences) and stained for IFN-γ (564336: BD Biosciences) and IL-2 (560547: BD Biosciences). All staining antibodies were used at 1:200 or 1:300 or 1:500 dilution depending on the experiment. Sample acquisition was performed on a BD Fortessa flow cytometer using FACS Diva software followed by data analysis with FlowJo software.