FC PROTOCOL

By admin

Flow cytometry (FC) is a technique for the rapid quantitative analysis and sorting of cells or other biological particles (e.g., microspheres, bacteria, small model organisms, etc.) in a liquid stream, one at a time, in a single row.

Intracellular Staining Procedure

1. Preparation of single cell suspensions;

2. PBS was washed twice before adding fixative;

(1) 4% PFA was added when the target was intracellular for 10-20 min of fixation and washed 1-2 times with flow cytometry perm buffer (5 min each time, 400-600 g);

(2) Transcription factor staining buffers were added when the target was in the nucleus. (Fixation for 30-60 minutes, washed 1-2 times for 5 minutes each time, 400-600 g);

3. After washing, add the appropriate buffer and allow the cells to resuspend for 10 minutes;

4.[Optionally, block Fc receptors (10-20 min);

5. Add antibody and incubate at 2-8°C (30 min);

6. Wash 1-2 times with appropriate buffer (5 min each, 400-600 g);

7. If non-fluorescent dye direct labeled antibody, add fluorescent secondary antibody and incubate at 2-8°C (30 min);

8. Wash 1-2 times (5 min each time, 400-600 g) with the appropriate buffer;

9. [Optionally, stain cells with cell reactive dyes;

10. Resuspend the cells with the appropriate buffer (or PBS);

11. Analyzing the cells by flow cytometry.

Cell Surface Staining Procedure

1. Prepare single cell suspensions;

2. [Optionally, block Fc receptor (10-20 min);

3. Add antibody and incubate at 2-8°C (30 min);

4. Wash with flow cytometric staining buffer (or PBS) (1-2x5 min, 350g);

5. If non-fluorescent dye direct labeled antibody, add fluorescent secondary antibody and incubate at 2-8°C (30 min);

6. Wash with Flow Cytometry Staining Buffer (1-2x5 min, 350g);

7. [Optionally, stain cells with cell reactive dyes;

8. Resuspend cells with Flow Cytometry Staining Buffer (or PBS);

9. Flow cytometric analysis of cells.

10X PBS 1000 ml, KH2PO4 2.4 g, NaH2PO4·12H2O 37.2 g, NaCl 80 g, KCL 2 g, ddH2O 1000 ml, pH 7.2-7.4.