Staining Methods for cell death
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<center> <h1> <font>Staining Methods for cell death Z. Xia 10/2/95</font></h1> </center>
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The simplest way: trypan blue.
Dead cells stain blue -
Non-fixed cells: FDA(fluorescein diacetate)-green, alive cells;
P.I. (propidium iodide)-red, dead cells35 mm plates:
- 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.
- 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)
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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.
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P. I.: Sigma, dissolve in PBS
FDA: Sigma, dissolve in acetone
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P. I. staining for fixed cells
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Fixation:
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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
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ETOH fixation-gives brighter P.I. staining
- Aspirate off media
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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:- 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.
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Fixation:
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Hoescht staining
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Fix cells
- remove media, fix w/ 4% paraformaldehyde/4%sucrose in PBS, neutral pH, RT 15-45 min
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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
- 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.
Morphologically:
Alive cells: phase brightNecrotic: 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.
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 � -
Fix cells