Cd8 pe cyanine7

Evaluation of T cell proliferation in immunized mice was conducted using a FACSCalibur flow cytometer (BD Biosciences, Milpitas, CA, USA). Briefly, a total of 1,000,000 mouse splenocytes were stimulated with the SARS-CoV-2 RBD peptides pool (4 μg/mL of each peptide) for 4 h at 37 °C with 5% CO₂. Brefeldin A (1 mg/mL, BD Sciences, Milpitas, CA, USA) was, then, added to the splenocytes and incubated for an additional 4 h. The splenocytes were washed twice with the PBS, and then stained with fluorescently conjugated antibodies to CD3-FITC (BD Pharmingen, San Diego, CA, USA), CD4 (PerCP-Cyanine5.5, BD Pharmingen, San Diego, CA, USA), CD8-PE-Cyanine7 (BD Pharmingen, San Diego, CA, USA), CD44-APC (BD Pharmingen, San Diego, CA, USA), and CD62L-PE (BD Pharmingen, San Diego, CA, USA). Zombie NIR™ Fixable Viability Kit (BioLegend, San Diego, CA, USA), whose stain has similar emission to APC/Cy7, was used to evaluate live or dead status of mammalian cells. Data were analyzed using FlowJo software (Version 10.8.1).

Cells were stained with CD3 FITC (Cat# 11-0039), CD3 eFluor 450 (Cat# 48-0038), CD4 PE-Cyanine7 (Cat# 25-0049), CD4 PerCP-Cyanine5.5 (Cat# 560650, BD Pharmingen), Foxp3 APC (Cat# 17-4777), Foxp3 PE (Cat# 12-4776), CD45RA PE (Cat# 130-092-248, Miltenyi), CD45RA APC (Cat# 130-092-249, Miltenyi), CD62L APC (Cat# 17-0629), CD25 PE (Cat# 130-091-024, Miltenyi), CD25 APC-H7 (Cat# 560225, BD Pharmingen), CD8 PE-Cyanine7 (Cat# 25-0088), Perforin PE (Cat# 12-9994), granzyme B PE (Cat# 12-8899), CCR8 APC (Cat# FAB1429A, R&D System), CD4 FITC (Cat# 11-0049), CD45 APC (Cat# 17-9459), CD14 PE (Cat# 12-0149), CD45RO PE (Cat# 12-0457), CD45RO eFluor 450 (Cat# 48-0457), CD127 APC (Cat# 17-1278). PITPNM3 antibody (Cat# NBP1-31070, Novus) was labeled with APC by Abcam APC Conjugation Kit (ab201807, Abcam) according to the manufacturer's instructions. For the intracellular stain, cells were pretreated with Intracellular Fixation and Permeabilization kit (Cat# 88-8824) according to the manufacturer's instructions. All the reagents were from eBioscience unless indicated otherwise. Cells were subsequently analyzed by multicolor flow cytometry (Gallios, Beckman Coulter, China).