Mouse anti chicken cd4 pe cy7

Eighteen hours post-treatment, mononuclear cells were stained with 7-AAD and subsequently fixed for 30 min using the fixation/permeabilization kit (BD Bioscience, Mississauga, ON, Canada). Cells were incubated in fixation and permeabilization buffer for 30 min at 4°C, blocked (20 min at 4°C) with 1% bovine serum albumin (BSA) followed by incubation (20 min at 4°C) with anti-cIFN-γ-biotin (ThermoFisher Scientific, Canada). Cells were washed twice in staining buffer (1% BSA) and stained with streptavidin-APC (ThermoFisher Scientific, Canada) for 20 min at 4°C in staining buffer. Mononuclear cells were washed twice in staining buffer and incubated with a mouse anti-chicken CD3ζ-PB, mouse anti-chicken CD4-PE-CY7 or mouse anti-chicken CD25-PE-CY7, mouse anti-chicken CD8α-FITC, and mouse anti-chicken γδTCR-PE (Southern Biotech, Canada). All cells were acquired on a BD FACS Canto II, and the data were processed by FlowJo V10 software.

Peripheral blood samples were collected from the wing veins of 10 birds in each group at 0, 7, 14, 21 and 28 dpv. Peripheral blood lymphocytes were isolated from the peripheral blood samples. The samples were diluted with equal volumes of sterile PBS, overlaid onto chicken peripheral blood lymphocyte isolation liquid (Solarbio, Beijing, China) and centrifuged at 500 g for 20 min at room temperature. The peripheral blood lymphocytes at the interface were collected and washed with sterile PBS. A final volume of 0.1 mL was stained with 3 µL of Mouse Anti-Chicken CD3-PE, 2 µL of Mouse Anti-Chicken CD4-PE/CY7 and 2 µL of Mouse Anti-Chicken CD8α-FITC antibodies (SouthernBiotech, Birmingham, AL, USA), respectively. The concentrations of the Mouse Anti-Chicken CD3-PE, CD4-PE/CY7 and CD8α-FITC antibodies were adjusted to 0.3, 0.1, and 1 µg/0.1 mL, respectively. All the samples were assessed for the percentages of CD3⁺, CD4⁺ and CD8⁺ T lymphocytes using a BD AccuriTMC6 Flow Cytometer (Becton, Dickinson and Company, Minneapolis, MN, USA). All the staining reactions were conducted according to the manufacturer’s protocols.

The antibodies mouse anti-chicken KULO1-FITC, mouse anti-chicken MHC II-PE, mouse anti-chicken CD3_ζ-PB, mouse anti-chicken CD4-PE-CY7, mouse anti-chicken CD8α-FITC, and mouse anti-chicken γδTCR-PE were purchased from Southern biotech, Canada. The following mouse anti-chicken CD25-APC (Biorad, Canada) and mouse anti-chicken IFN-γ biotin and streptavidin APC were purchased from Life Technologies, Canada.