35 mm plates:

1. to 2 ml medium or PBS, add 2 ul 2 mg/ml P. I. 6 ul 5 mg/ml FDA
2. R. T. 3 min
3. Rinse 1 X PBS
4. leave cells in PBS. Examine cells under the scope immediately.

1. 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
2. After staining, need to examine the staining right away, otherwise, the green staining gets diffused. You can leave cells at 4 ¡C for a few hr.-overnight to slow down the diffusion (I have tried 3T3, do not know if it works for neurons)
3. Ref.: K. H. Jones & J. A. Senft (1985) J. Histochemistry & Cytochemistry 33: 77-79
   M. Schramm et al., (1990) PNAS 87: 1193-1197
4. This method stains for non-fixed cells.
5. P. I.: Sigma, dissolve in PBS
   FDA: Sigma, dissolve in acetone

1. Fixation:
   1. ETOH fixation-gives brighter P.I. staining
      Gently overlay over media 4X media vol. of ETOH precooled to -20 ¡C
      R.T. 3 min
      Gently mix media & ETOH with pipet
      R.T. 5 min
      or
   2. 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
2. Aspirate off media
3. 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:
   1. 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.
   2. 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.

1. Fix cells
   1. remove media, fix w/ 4% paraformaldehyde/4%sucrose in PBS, neutral pH, RT 15-45 min
   2. 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
2. wash 1X PBS/0.1 % triton X-100, RT 5 min
3. stain cells w/ 2.5 ug/ul Hoeschst 33258 in PBS/0.1 % triton X-100 R.T. 5 min
4. wash 1X PBS/0.1 % triton X-100, RT 5 min
5. Mount w/ coverslips. Examine cells uder fluorescence scope using DAPI filter

1. Alive cells should have evenly stained nuclei. Nuclei from apoptotic cells show condensed, or fragmented morphology.
2. Hoeschst 33258, Sigma B-2883 (bis-Benzimide), 5 mg/ml in H2O stock. Light sensitive.
3. Hoeschst 33258 stains permeablized cells; Hoeschst 33342 is permable, can stain both fixed and non-fixed 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:

1. Trypan blue: dead cells stain blue. Can not distinguish necrotic vs terminally apoptotic cells
2. FDA/P.I. staining: Alive cells stain blue, necrotic or terminally apoptotic cells stain red. Early apoptotic cells should not stain red.
3. P. I. or Hoeschst staining of fixed cells: Nuclei from apoptotic cells show condensed, or fragmented morphology.
4. Tunnel staining: commercial kits available. Nuclei from apoptotic cells show condensed, or fragmented morphology.
5. DNA ladder: Necrotic cells do not show DNA laddering; Most, but not all, of the apoptotic cells show DNA laddering.