Approximately 1 × 106 cells were stained with 5–10 μg/ml of CD45-Pacific Blue or 2.5 μl of CD71 stock solution for 30 min at 4–8 °C in the dark. Cells were washed twice with PBS by centrifugation at 1455×g for 4 min, at 4 °C. After the last wash 200 μl of FACS buffer was added to the cells.
Double staining with SYBR Green I and CD45-Pacific Blue: A volume of 30 μl of SYBR Green I at 10× was added to pelleted cells that were previously stained with CD45-Pacific Blue (as mentioned above) for 30 min at 4–8 °C, in the dark. After incubation a volume of 200 μl of FACS buffer was added.
Triple staining with SYBR Green I, CD45-Pacific Blue and CD71-APC: Approximately 1 × 106 cells were stained with 10 μg/ml of CD45-Pacific Blue, 2.5 μl of CD71 stock solution and 30 μl of SYBR Green I at 10x. Samples were incubated for 30 min in the fridge (4–8 °C) in the dark. Cells were washed twice with PBS by centrifugation at 1455×g for 4 min, at 4ºC. After incubation a volume of 200 μl of FACS buffer was added.
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