The simplest way: trypan blue.
Dead cells stain blue
Non-fixed cells: FDA(fluorescein diacetate)-green, alive cells;
P.I. (propidium iodide)-red, dead cells
35 mm plates:
Note:
If the P.I. staining is not strong enough to be picked up easily under your scope, use 2 X P. I., i.e., 4 ul 2 mg/ml in 2 ml medium
After staining, need to examine the staining right away, otherwise, the green staining gets diffused. You can leave cells at 4 for a few hr.-overnight to slow down the diffusion (I have tried 3T3, do not know if it works for neurons)
Ref.: K. H. Jones & J. A. Senft (1985) J. Histochemistry & Cytochemistry 33: 77-79
M. Schramm et al., (1990) PNAS 87: 1193-1197
This method stains for non-fixed cells.
P. I.: Sigma, dissolve in PBS
FDA: Sigma, dissolve in acetone
to 2 ml medium or PBS, add 2 ul 2 mg/ml P. I. 6 ul 5 mg/ml FDA
R. T. 3 min
Rinse 1 X PBS
leave cells in PBS. Examine cells under the scope immediately.
P. I. staining for fixed cells
P. I. will stain for both DNA and RNA. It is critical to include RNase A to eliminate the cytosolic RNA staining background. If use ETOH fixation, it is less critical to include RNaseA in staining soln.
This will stain both alive and dead cells. Alive cells should have evenly stained nuclei. Nuclei from apoptotic cells show condensed, or fragmented morphology. Can not distinguish necrosis.
ETOH fixation-gives brighter P.I. staining
Gently overlay over media 4X media vol. of ETOH precooled to -20
R.T. 3 min
Gently mix media & ETOH with pipet
R.T. 5 min
or
Paraformaldehyde fixation: (8 % paraformaldehyde/4% sucrose/ in PBS, pH 7.2-7.6)
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
Fixation:
Aspirate off media
Staining:
4 ug/ul P. I./0.1 % triton X-100/0.5 mg/ml RNaseA in PBS
R.T.5 min
Examine under the scope or mount with coverslips Note:
Hoescht stainingNote: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:
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
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.
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.
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
Fix cells
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
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