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Staining of cell surface

Cell preparation:
1. Collect tissues(peizang, lymph nodes, thymus and bone marrow), and then make single cell suspension by cell staining buffer. For vitro stimulated cells, suspend the stimulated cells in cell staining buffer directly.
2.Add cell staining buffer to 15 mL, then cell suspension is centrifuged at 350×g for 5 minutes, discard the supernatant.

Erythrocyte lysis:
3. If it needs to lyse erythrocyte (e.g. peizang), dilute 10× erythrocyte lysis buffer (RBC) to 1×RBC with H2O at first, then place 1×RBC in room temperatures. re-suspend the cells in 3 mL 1×RBC, and incubate cells for 5 minutes at room temperature.
4.Add 10 mL cell staining buffer to terminate erythrocyte lysis, then cell suspension is centrifuged at 350×g for 5 minutes, discard the supernatant.
5. Repeat washing once like step 2.
6. Cell counting, cells were prepared to 1 × 107 cells/mL of cell suspension by cell staining buffer. Add 100μl cell suspension into flow tube for standby.

Enclosing of Fc receptor:
7.Enclosing Fc receptor can reduce non-specific staining during staining process. In the mouse, purified CD16/CD32 monoclonal antibody can bind with FcγRⅢ/Ⅱ, and block nonspecific staining. It needs to add 5-10 μg/mL Fc γ RⅢ/Ⅱ blocking solution, and incubate on ice for 10 minutes.

Cell staining:
8. Fluorescently labeled antibody is added into cells in recommended dosage of instruction, mix evenly, then place on ice, and incubate cell suspension in dark for 30 minutes.
9.Add 5mL FACs buffer to re-suspend cells, the cell suspension is centrifuged at 350×g for 5 minutes, discard the supernatant.
10.Add 0.5mL FACs buffer to re-suspend cells, and start to detect and analyze by flow cytometry.

Notice:
1.Before antibody is used, it is recommended to swing the tube one time quickly, in order to make antibody assemble in the bottom of the tube;
2.Fluorescently labeled antibody should be stored at 4℃ in the dark, do not freeze them;
3.Once the samples are fixed or dyed with paraformaldehyde, it is suggested to detect as soon as possible, delayed analysis may reduce fluorescent signal of certain antibody.
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