Live dead fixable red dead cell kit

Cells were detached with trypsin, and sequentially stained with primary and secondary antibody (unless the primary antibody was fluorescently labeled) in FACS staining buffer (PBS + 0.5% BSA + 0.02% sodium azide) for 1 h at 4 °C. Cells were washed three times with FACS buffer after incubation with primary and secondary antibody, and analyzed using FACSCalibur (BD Biosciences) and FlowJo software (Tree Star). Staining with propidium iodide was carried out to distinguish dead from live cells in unfixed cells, and the Live/Dead Fixable Red Dead Cell kit (L34971, Invitrogen) was used in experiments that required fixing and intracellular staining. To distinguish transfected vs untransfected cells, intracellular staining for the protein of interest was carried out after surface staining for MHC-I. Cells were fixed with 4% PFA (or CytofixCytoperm for virus infected cells), followed by cell permeabilization with 0.4% TritonX-100 in in intracellular FACS buffer (TBS + 2.5% BSA +0.1% Tween20), followed by staining with antibodies in intracellular FACS buffer overnight at 4 °C. Cells were then washed with TBS followed by staining with secondary antibodies or analysis as mentioned above.

1Cells were detached with trypsin and sequentially stained with primary and secondary antibodies (unless the primary antibody was fluorescently labeled) in FACS staining buffer (PBS + 0.5% BSA + 0.02% sodium azide) for 1 h at 4 °C. Cells were washed three times with FACS buffer after incubation with primary and secondary antibodies and analyzed using FACSCalibur (BD Biosciences) and FlowJo software (Tree Star). Staining with propidium iodide was carried out to distinguish dead from live cells in unfixed cells, and the Live/Dead Fixable Red Dead Cell kit (L34971, Invitrogen) was used in experiments that required fixing and intracellular staining. To distinguish transfected vs. untransfected cells, intracellular staining for the protein of interest was carried out after surface staining for MHC-I. Cells were fixed with 4% PFA (or CytofixCytoperm for virus-infected cells), followed by cell permeabilization with 0.4% TritonX-100 in in intracellular FACS buffer (TBS + 2.5% BSA + 0.1% Tween20), followed by staining with antibodies in intracellular FACS buffer overnight at 4 °C. Cells were then washed with TBS followed by staining with secondary antibodies or analysis as mentioned above.