Question: I would like to culture neuron cells and detect apoptosis using FAM FLICA kits. I checked the adherent protocol for staining but it seemed for suspension cells. I will perform on 24 well plate and use a microscope. Could you direct me to a detailed and precise protocol?
Thanks for your interest in our FAM FLICA Assay! You may find the manual instructions to be somewhat general for cellular staining. Here’s a brief protocol that is helpful.
- If staining adherent cells for microscopy analysis, we recommend adding FLICA at 1X working strength directly to the overlay medium for staining.
- Reconstitute FLICA vial with 50 µL DMSO to form a 150-300X stock concentrate.
- Dilute FLICA 1:5 to form a 30-60X working solution.
- When ready for FLICA staining, spike 30-60X working solution into the overlay medium in your wells at 1X staining concentration. For example, if the final well volume is 300 µL, add 5-10 µL of 30-60X FLICA per well.
- Stain for 30 minutes to several hours (2-3 hours is fine).
- Carefully remove overlay medium containing FLICA and replace with wash buffer or fresh culture medium; incubate 10 minutes at 37°C; repeat wash steps 2 additional times. The entire plate may be gently spun as part of the wash process to sediment any loose floating cells.
- Adherent cells need to be carefully washed to avoid the loss of any apoptotic cells that round up and come off the plate surface. If you are unable to centrifuge the entire plate, we recommend saving and pooling overlay media (containing FLICA stain) and wash buffer (from all 3 wash steps). Pellet to recover any loose cells via centrifugation. Remove supernatant and resuspend in wash buffer or fresh cell culture medium. Repeat centrifugation/resuspension wash steps 2 more times (pellet, remove supernatant, and resuspend). These cells can be recombined with your adherent samples or analyzed separately according to your work flow.
- Carry out any counterstaining steps with compatible ancillary dyes.
- Image your samples.
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