Staining Methods for cell death

http://www.microimage.com.cncyh(2010-11-24 12:00:16)

  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:

  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. 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

  P. I. staining for fixed cells

  Fixation:

  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

  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:

  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.

  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 

  

更多相关搜索: