Mouse RAW264.7 Cell Polarization Induction Kit I

Recommended Protocol:

Cell Line: RAW264.7

I.M1 Polarization

Culture Conditions:6-well cell culture plate, 2*10^5 cells per well, adhered for 24h. Replace the culture medium with2%FBS+DMEM(to minimize serum factor interference), add the corresponding cytokines according to the table below, and perform flow cytometry detection after 24h.

Flow Cytometry Protocol:

1. Cell Harvesting: After 24h of cytokine treatment, discard the culture medium supernatant, wash cells once with PBS, discard the supernatant, then gently pipette the adherent cells with PBS to resuspend; collect cells by centrifugation.

2. Cell Counting: Resuspend cells in 0.5ml of 1%BSA, calculate the total cell number, and check cell viability, which must be >95%.

3. Antibody Incubation: Add PE Rat anti-Mouse CD86 mAb (A27137) (refer to the manual for antibody dosage) or an isotype control , incubate at room temperature for30min .

4. Cell Washing: Wash cells with 1%BSA to remove residual antibodies, resuspend cells in 1%BSA.

5. Acquire data.

II. M2 Polarization

Culture Conditions:6-well cell culture plate, 2*10^5 cells per well, adhered for 24h. Replace the culture medium with2%FBS+DMEM(to minimize serum factor interference), add the corresponding cytokines according to the table below, and perform flow cytometry detection after 24h.

Flow Cytometry Protocol:

1. Cell Harvesting: After 24h of cytokine treatment, discard the culture medium supernatant, wash cells once with PBS, discard the supernatant, then gently pipette the adherent cells with PBS to resuspend; collect cells by centrifugation.

2. Cell Counting: Resuspend cells in 0.5ml of 1%BSA, calculate the total cell number, and check cell viability, which must be >95%.

3. Fixation: Collect cells by centrifugation, discard the supernatant, resuspend and fix cells in 0.2mL 4% paraformaldehyde at room temperature for 15min, then wash cells, centrifuge and discard the supernatant (to remove residual fixative).

4. Permeabilization: Resuspend cells in 0.2ml 1X permeabilization buffer for permeabilization at room temperature for 30min.

5. Blocking: Block cells in each well with 2μg CD16/CD32 antibody for blocking, incubate on ice for30min.

5. Antibody Incubation: Add APC anti-Mouse CD206 (MMR) mAb (A26785) (refer to the manual for antibody dosage) or an isotype control APC Rat IgG Isotype Ctrl Antibody), incubate at room temperature for 30min .

6. Cell Washing: Wash cells with 1%BSA to remove residual antibodies, resuspend cells in1%BSA.

7. Acquire data.