Hoescht staining - Fix cells
- remove media, fix w/ 4% paraformaldehyde/4%sucrose in PBS, neutral pH, RT 15-45 min
- if cells are not adhereing well to the plates:
Gently overlay over media 2X media vol. of 8 % paraformaldehyde/4% sucrose/ in PBS, pH 7.2-7.6
gently tilt the plates to mix
R.T. 15 min
- wash 1X PBS/0.1 % triton X-100, RT 5 min
- stain cells w/ 2.5 ug/ul Hoeschst 33258 in PBS/0.1 % triton X-100 R.T. 5 min
- wash 1X PBS/0.1 % triton X-100, RT 5 min
- Mount w/ coverslips. Examine cells uder fluorescence scope using DAPI filter
Note: - Alive cells should have evenly stained nuclei. Nuclei from apoptotic cells show condensed, or fragmented morphology.
- Hoeschst 33258, Sigma B-2883 (bis-Benzimide), 5 mg/ml in H2O stock. Light sensitive.
- Hoeschst 33258 stains permeablized cells; Hoeschst 33342 is permable, can stain both fixed and non-fixed cells.
To distinguish alive vs necrotic, apoptotic cells: Morphologically:
Alive cells: phase bright
Necrotic: cell swelling, i.e., enlarged cell bodies, cell membrane leakage, lysis of cell body
Apoptotic: rough membrane, plasma membrane shrinkage, cell body shrinkage, membrane blebbing, no lysis of cell body
Staining:
- Trypan blue: dead cells stain blue. Can not distinguish necrotic vs terminally apoptotic cells
- FDA/P.I. staining: Alive cells stain blue, necrotic or terminally apoptotic cells stain red. Early apoptotic cells should not stain red.
- P. I. or Hoeschst staining of fixed cells: Nuclei from apoptotic cells show condensed, or fragmented morphology.
- Tunnel staining: commercial kits available. Nuclei from apoptotic cells show condensed, or fragmented morphology.
- DNA ladder: Necrotic cells do not show DNA laddering; Most, but not all, of the apoptotic cells show DNA laddering.
Positive control for apoptosis:
1 uM staurosporin in media, 3-24 hr for most of the cells, always induces apoptosis (as far as we know). staurosporin: 1 mM stock in DMSO, 4 ¡C