Cytofix and perm

Cells harvested from overnight stimulation cultures were incubated with live/dead aqua V510 for 15 min on ice. The cells were then surface stained for 30 min on ice using anti-CD3-FITC (BioLegend, clone UCHT1, 1:200, Cat# 300406), anti-CD4-APC-H7 (BD Pharmingen™, clone RPA-T4, 1:200, Cat# 560158), and anti-CD8-PerCPCy5.5 (BD Biosciences, clone RPA-T8, 1:200, Cat# 560662) antibodies. After fixation and permeabilization with Cytofix and Perm (BD Biosciences, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min using anti-TFNα-PE-Cy7 (BD, clone MAb11, 1:200, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, 1:200, Cat# 554702) antibodies. After the final wash step, the cells were resuspended in 200 µL of FACS buffer. Samples were acquired using an FACSAria III instrument (BD Biosciences, San Diego, CA, USA) and analysed using the FlowJo software (Treestar, San Carlos, CA, USA).

Cells harvested from the overnight or 10-day stimulation cultures were washed and incubated with Live/dead aqua V510 for 15 min on ice. Cells were then washed again and surface-stained for 30 min on ice with the following antibodies: anti-CD3-FITC (BioLegend, clone UCHT1, 1:200, Cat# 300406), anti-CD4-APC-Cy7 (BD Pharmingen™, clone RPA-T4, 1:200, Cat# 561839), anti-CD8-PerCPCy5.5 (BD Bioscience, clone RPA-T8, 1:200, Cat# 560662). After fixation and permeabilization with Cytofix and Perm (BD Bioscience, Cat# 554714) on ice for 15 min, intracellular staining (ICS) was performed on ice for 30 min with anti-TNF-PE-Cy7 (BD, clone MAb11, 1:200, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, 1:200, Cat# 554702). After the final wash, cells were resuspended in 200 µl FACS buffer. The samples were acquired using an FACSAria III instrument (BD Bioscience) and analyzed with FlowJo software (Treestar).

In total, 0.5–1 × 10⁶ cells were harvested from the 16-h or 10-day stimulation cultures, washed, and incubated with Live/Dead Aqua V510 for 15 min on ice. The cells were then washed again and surface-stained for 30 min on ice with the following antibodies: anti-CD3-FITC (BioLegend, clone UCHT1, Cat# 561802), anti-CD4-BV421 (BioLegend, clone OKT4, Cat# 317434), anti-CD8-PerCPCy5.5 (BD Pharmingen™, clone RPA-T8, Cat# 560662), anti-CD27-PE (BD Pharmingen™, clone M-T271, Cat# 555441), and anti-CD45RA-APC-H7 (BD Pharmingen™, clone HI100, Cat# 560674). After fixation and permeabilization with Cytofix and Perm (BD Bioscience, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min with anti-TNF-PE-Cy7 (BD, clone MAb11, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, Cat# 554702). After the final wash, the cells were resuspended in 200 μL FACS buffer. A FACSFortessa instrument (BD Bioscience) was used to acquire data, which were analyzed using FlowJo software (Treestar).