General protocol for flow cytometry

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

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