1.1 Flow Cytometry Staining Protocol for Cell Surface

1 Single-cell staining  The process is as follows:
- Sample Source: Peripheral blood, bone marrow, tissue blocks, cultured cells, exfoliated cells, etc.
- Prepare a single-cell suspension.
- Add Reagents: Add fluorescent antibodies or fluorescent dyes at a concentration of 0.2 – 1 × 10⁶ cells/100μl.
- Incubation: Perform incubation.
- Centrifugation and Washing: Centrifuge, wash, and resuspend in PBS.
- Instrument Detection: For detection, the final volume is 0.5 – 1 ml, and the final concentration is 1×10⁶ cells/ml. The process is as follows:
- Sample Source: Peripheral blood, bone marrow, tissue blocks, cultured cells, exfoliated cells, etc.
- Prepare a single-cell suspension.
- Add Reagents: Add fluorescent antibodies or fluorescent dyes at a concentration of 0.2 – 1 × 10⁶ cells/100μl.
- Incubation: Perform incubation.
- Centrifugation and Washing: Centrifuge, wash, and resuspend in PBS.
- Instrument Detection: For detection, the final volume is 0.5 – 1 ml, and the final concentration is 1×10⁶ cells/ml.2 Whole blood staining 3
Example of Cell Surface Staining of Human Peripheral Blood Mononuclear Cells (PBMCs) and Suspension Cell Lines
 (1)  Prepare  PBMCs  in  CPT  tubes or  Ficoll  density layer centrifuge tubes, then resuspend  PBMCs  or cell line cells in    Pharmingen Stain Buffer (BSA)  or  Stain Buffer (FBS) .  (2)  Add  1ml  pre-cooled    Pharmingen Stain Buffer, 300g, 4 ° C  centrifuge  5  min per tube. Repeat  2  times. Adjust the cell final concentration to  107 cells/ml  with pre-cooled    Pharmingen Stain Buffer .  (3) Transfer  100 μl  cell suspension ( 106  cells) to  12 x 75mm  round-bottomed polypropylene tubes or round-bottomed microplate flow tubes.  (4)  Add an appropriate amount of specific surface antibody to each tube and incubate on ice for  20  min in the dark.  (5)  Wash cells  2  times with an appropriate amount of  Stain Buffer  (microplate  200 μl/well/time , test tube  1ml/tube/time ),  300g  Centrifuge  5  minutes. Carefully aspirate or pour out the supernatant. ( 6)  Tap test tube or microplate to mix cells well.  (7)  For indirect staining, add an appropriate amount of secondary antibody or streptomycin-labeled antibody to  100 μl Stain Buffer  according to instructions, and repeat steps  4  and  5 . (8)  Appropriate amount of  Stain Buffer  Resuspend cells (microplate  200 μl/well , test tube  0.5 ml/well ).
Remarks:
1. For the preparation of PBMCs, please refer to the instruction manual for Ficoll products or erythrocyte lysates.
2. To avoid non-specific staining, add FCR blockers before adding surface antibodies.