Cd4 pe cy7 gk1.5

Spleens and mesenteric lymph nodes were collected from the control, poly I:C-treated, ST7-treated, and polyI:C+ST7-treated mice. All tissues were ground using a Wheaton tissue grinder, and then a cell strainer was used to filter out the debris. Red blood cells were lysed in an RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Splenocytes and lymph node cells were then suspended in a Roswell Park Memorial Institute (RPMI) culture medium containing 2% fetal bovine serum. To minimize the nonspecific binding of antibodies to FcR-bearing cells, a 2.4G2 hybridoma supernatant was used to pretreat these splenocytes and lymph node cells. After the 2.4G2 supernatant was removed by centrifugation, the cell surface molecules were stained with specific antibodies for 10 min and then washed twice with a staining buffer (1× PBS, 2% horse serum, and 0.05% NaN₃). Finally, all cells were resuspended in a staining buffer and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). The following antibodies were used in conjugation with a fluorochrome from BioLegend: CD3-FITC (2C11), CD8α-APC-Cy7 (53-6.7), CD19-PB (6D5), CD4-PE-Cy7 (GK1.5), and CD69-PE (H1.2F3). Positive cells were validated through comparison with the no-antibody staining control.

CD45 Alexa 700 (30-F11, Biolegend), CD19 PerCP/Cy5.5 (1D3/CD19, Biolegend), CD3ε Brilliant Violet 510 (17A2, Biolegend), CD4 PE/Cy7 (GK1.5, Biolegend), CD49b BV421 (DX5, Biolegend), CD8a Brilliant Brilliant Violet 711 (53–6.7, Biolegend), FoxP3 Alexa 647 (MF-14, Biolegend), and CD285 PE (TLR-5) (ACT5, BD Biosciences) (Supplementary Table 11).