1.1 Cell Surface Staining

1 Single-cell staining

• 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

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, centrifuge at 300g, 4 °C for 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), centrifuge at 300g for 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 into 100 μl Stain Buffer according to instructions, then repeat steps 4 and 5.
8. Resuspend cells with appropriate amount of Stain Buffer (microplate: 200 μl/well; test tube: 0.5 ml/tube).