Fixed/permeabilized cultured cells - BD Pathway Bioimager 855 (JUMC)

From LipidomicsWiki

Visualization of fixed/permeabilized cultured cells by BD Pathway Bioimager 855

Cell types: HUVECs, skin fibroblasts

1. Seed cells in 96-well clear bottom tissue culture plates for imaging applications (Becton Dickinson; Cat. No. 353119) at 10000 cells per well (starting recommendation)
2. Culture cells overnight (16 hours) in standard conditions to achieve 50% confluency (the initial number of cells per well and incubation time should be optimized empirically to account for specific growth rate differences between cell types so that the cell density optimal for image acquisition is not compromised)
3. Remove medium and wash cells with 100 µl PBS
4. Add 100 µl 1:1 v/v mixture of 100% methanol and 100% acetone (fixing/permeabilizating solution)
5. Incubate for 3 min at -20oC
6. Remove fixing/permeabilizating solution and dry cells (5—10 min)
7. Incubate in 100 µl PBS for 15 min
8. Add 100 µl PBS with 10% bovine serum albumin (BSA) and 1% Triton X-100 (blocking solution) and incubate for 1 h at room temperature
9. Remove blocking solution
10. Add primary antibodies diluted in 100 µl PBS with 10% BSA (specific antibody dilution must be determined empirically; manufacturer’s recommendations can be used as a starting guideline; typical range is between 1:100 and 1:1000)
11. Incubate overnight at room temperature
12. Remove solution
13. Incubate in 100 µl PBS for 10 min (repeat washing three times)
14. Add secondary antibody in 100 µl PBS with 10% BSA (secondary antibody is labeled with a fluorescent dye and must be have species specificity appropriate for the primary antibody)
15. Incubate for 1 h at room temperature and remove
16. Incubate in 100 µl PBS for 10 min (repeat washing three times)
17. Add Hoechst 33342 (Molecular Probes, Cat. No. H-3570) in 100 µl PBS (final concentration 2 ng/µl)
18. Incubate for 30 min at room temperature and remove
19. Incubate in 100 µl PBS for 10 min at room temperature and remove
20. Dry cells for 5—10 min
21. Image cells using the 20x/NA 0,75 objective and AttoVision software (select Hoechst for nuclear ROIs, and secondary antibody label for cytoplasmic ROIs, or as appropriate; avoid prolonged exposure to light to prevent bleaching)
22. If necessary for further use, store the plate in dark