Pe cy7 conjugated cd3

PBMCs (2 × 10⁶ cells/ml) were stimulated in RPMI 1640 culture medium containing 20 mMl‐glutamine (Gibco), 1% penicillin–streptomycin (Gibco), and 10% fetal bovine serum (batch no. F9665, lot no. 030M3399; Sigma) for 3 hours with phorbol myristate acetate (PMA) (50 ng/ml; Sigma) and ionomycin (750 ng/ml; Sigma) in the presence of GolgiStop (BD Biosciences), in accordance with the manufacturer's instructions. Surface staining for PE/Cy7‐conjugated CD3, APC/Cy7‐conjugated CD14 (BioLegend), and Pacific Blue–conjugated CD45RO was followed by intracellular staining for PerCP/Cy5.5‐conjugated CD4, PE‐conjugated IL‐17A (BioLegend), FITC‐conjugated TNF (BioLegend), or Alexa Fluor 647–conjugated FoxP3.

To measure the effects of RosA on virus-mediated apoptosis, both floating cells in the culture supernatant and adherent cells detached by EDTA-free trypsin were collected. After washing in 1× annexin binding buffer, cells in 100 µL of binding buffer were incubated with Annexin V-FITC and propidium iodide (PI) at room temperature in the dark for 30 min. Then, cells were analyzed by a flow cytometer (LSRII Fortessa; BD, San Jose, CA) within 4 h.
To analyze the levels of CD3⁺CD8⁺ T cells, 20 µL of peripheral blood sample from each group was preincubated with anti-FcγRIII/II (Fc block) (Anti-Mouse CD16/32; 553141). After 30 min of incubation, the following fluorochrome-labelled antibodies: PeCY-7-conjugated CD3 (Clone: 145-2C11; Biolegend), PE-conjugated CD4 (Clone: GK 1.5; Biolegend) and APC-conjugated CD8 (Clone: 53-6.7; Biolegend), were added to the samples for a further 30 min of incubation at room temperature. Afterward, ammonium-chloride-potassium (ACK) lysing buffer was used to lyse red blood cells in the blood samples. Finally, samples were resuspended in 500 µL of staining buffer and analyzed by a flow cytometer (LSRII Fortessa; BD, San Jose, CA).