I. Materials required
Reagents
- One (or more) Iaire antibody (s) coupled or not and specific to your molecule (s) of interest
- An Fc receptor blocker or “Anti-CD16 / 32, block Fc binding” which binds the primary antibody in a non-specific manner
- A secondary antibody, in the case of indirect labeling with an uncoupled primary Ab
- One (or more) control isotypes that are not related to isotypes and couplings identical to those of primary antibodies
Buffers
- A flow cytometry marking buffer or “Staining Buffer”
- Trypan blue for cell counting or “Trypan blue”
- Sheath liquid for the flow cytometer
Equipment – Pipettes and a Pipet-aid
- A centrifuge
- A refrigerator or ice
- A flow cytometer
II. Duration of the experiment
- 30 minutes of sample and antibody preparation (dilutions)
- 30 min to 1 h of incubation of the antibodies depending on the strategy used (direct or indirect labeling)
- 30 minutes to 1 hour of cell reading by the flow cytometer (depending on the number of samples, the concentration of the cells and the settings of the cytometer)
- TOTAL: 2 to 3 hours
III. Procedure
- Dilute the primary antibody and the control isotype in 50 μl of reaction buffer in order to obtain the desired concentration and validated beforehand by a titration (identical for the two Ab)
- Incubate 50 µl of the cell preparation with 50 µl of the preparation of Iaire antibody or control isotype in a 96-well U-bottom plate
- Gently shake the plate and incubate for 15-30 minutes at 4 ° C and in the dark
Note: Antibodies have different affinities with their antigen and require variable incubation times to be optimized beforehand.
- Add 200 µl of reaction buffer to stop the reaction
- Centrifuge the cell suspension for 5 min (300 – 400g) at + 4 ° C and remove the supernatant.
- Repeat the washing of the cells twice with 200 μl of reaction buffer and centrifugation of the cell suspension for 5 min (300 – 400g) at 4 ° C
Optional: In case of indirect labeling, repeat the labeling steps with the secondary antibody with the same protocol and the same observations
- Resuspend in 500 µl of reaction buffer or fixation buffer supplemented with 2% formaldehyde
- Read the cells marked with the flow cytometer
IV. Search engines
- Primary antibodies
- Secondary antibodies, in the case of indirect labeling with an uncoupled Ac Iaire
