Fitc conjugated anti mouse cd4 antibody

Collected blood (100 uL) was stained with 20 uL of PE-conjugated anti-mouse CD8 and FITC- conjugated anti-mouse CD4 antibodies (Biolegend, San Diego, CA, USA) for 1 hr in a dark room. Blood was stirred softly and incubated for 30 min in a cold dark room. Blood was mixed with 500 uL of OptiLyse® C no-wash lysing solution (Beckman Coulter Inc., Indianapolis, IN, USA) and incubated for 10 min in a cold dark room. The sample was centrifuged at 1,800 rpm for 5 min and the cell pellets were washed thrice using FACS buffer (PBS containing 0.05% NaN₃, 5% FBS, pH 7.2) and centrifuged. The isolated pellet was added to 0.5 mL FACS buffer and was acquired on a BD FACS Aria™ III (BD Biosciences, Franklin Lakes, NJ, USA). Splenocyte (4x10⁶cells) was mixed with 200 uL FACS buffer (1XPBS, 5% FBS, 0.05% NaN₃) and incubated for 30 min. The cells were stained with 20 uL of PE-Cy5 conjugated anti-mouse CD3, PE- conjugated anti-mouse CD8, and FITC- conjugated anti-mouse CD4 antibodies (Biolegend, San Diego, CA, USA) for 30 min in a cold dark room and then the cells were centrifuged at 1,500 rpm for 5 min. The cells were washed twice with FACS buffer, suspended in 250 uL PBS containing 1% formalin, and then acquired on a BD FACS Aria™ III.

CD4⁺ T cells were labeled with FITC-conjugated anti-mouse CD4⁺ antibodies (Biolegend) according to the manufacturer’s instructions. These cells were then stained with fluorescein-isothiocyanate (FITC)-labeled anti-mouse IFN-γ monoclonal antibody (Biolegend), and PE-labeled anti-mouse IL-4 monoclonal antibody (Biolegend) for 30 min on ice. Flow cytometric analysis was performed in a BD LSRII based on BD FACSDiva software (Becton Dickinson, CA, USA).

CD4⁺ T cells found among the isolated splenocytes were labeled with fluorescein-isothiocyanate (FITC)-conjugated anti-mouse CD4⁺ antibodies (Biolegend) and stained with phycoerythrin (PE)-labeled anti-mouse interferon-γ (IFN-γ) or IL-4 monoclonal antibodies (Biolegend) for 30 min at a cold temperature. Flow cytometric analysis was performed with FlowJo software (FlowJo LLC, Ashland, Oregon).