Flow cytometry was performed to characterize cell populations present in samples collected during the apheresis process. A combination of stains and antibodies were used to identify cells containing DNA/RNA (SYBR Green I), white blood cells (WBCs) (CD45 antibody) and/or reticulocytes (CD71 antibody). Samples from subject 1 were stained with SYBR Green I (Molecular Probes); samples from subjects 2 and 3 were stained with SYBR Green I and CD45-PacificBlue; and samples from subject 4 were stained with SYBR Green I, CD45-Pacific Blue and/or CD71-APC. Samples were kept on ice or at 4–8 °C until analysed by flow cytometry.

A volume of 2.5 μl or 1 × 106 cells from each sample was stained with 30–50 μl of SYBR Green I at 10× for 30 min in the dark. After incubation, 200 μl of FACS buffer (2% fetal bovine serum in phosphate buffered saline) was added.

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.

Samples from subjects 1, 2 and 3 were acquired on a FACS CANTO II (BD Biosciences), using the 488 nm and 405 nm lasers. SYBR Green I positive cells were detected using a 530/30 nm band-pass filter and CD45-Pacific Blue positive cells were detected using a 450/50 nm band-pass filter. Samples from subject 4 were acquired on a LSR FORTESSA (BD Biosciences), using the 488 nm, 640 nm and 405 nm lasers. SYBR Green I positive cells were detected using a 530/30 nm filter, CD45-Pacific Blue positive cells were detected using a 450/50 nm filter and CD71-APC positive cells were detected using a 670/14 nm filter. Flow cytometry data was analysed using FlowJo® software (version 10.8, Tree Star Inc, Oregon, USA).

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