LSC –sample preparation protocol

Sample preparation:

• Seed cells on slides and allow to grow to 50-80% confluence

• Fix cells by dipping them into icecold Methanol and incubate >20 min at –20’C (or overnight; for other fixatives see additional notes).

Staining:

• Rehydrate samples in PBS for 5 min
• Wash 5 min in PBS+0.05% triton-X100 (PBS-TX) to break down membrane barriers
• Incubate in 200 mg/ml Rnase + 5 mg/ml PI in PBS-TX for 45 min – several hours at 37’C
• Rinse in PBS
• Embed in antifading reagent + PI (0.5 mg/ml) and seal with nailpolish
• Store slides at 4’C in the dark

In combination with immunofluoresence:

• Post-stain with PI-RNase or add PI-Rnase to antibody mixes and incubate at 37’C.

Notes:

Cells grown on slides versus coverslips:

Slides: remain better in focus during scanning (culture in petridishes or use Lab-Tek chamberedslides)

Coverslips: allow high-resolution imaging of same sample, but need to be refocused regularly

Refocussing during scan:

• open scan-data window
• wait until scan is finished
• refocus by eye
• close window to resume scanning

Suspension cells:

Suspension cells and trypsinized cells can be cytospun to slides.

• Cytospin 10.000 - 20.000 cellen / spin (in ca 0.2 ml) to slide

• Airdry shortly
• fix

However, the DNA histogram appears less robust and mitotic cells cannot be recognized anymore in the scattergram of PI-max versus PI-integration. When interphase nuclei are regularly shaped with an overall equal staining, the mitotic population may be recognized in a scattergram of PI-intergation versus PI-texture.

Fixation:

Cells can probably be fixed in any fixative.

Keep an eye on preventing selective cell loss (e.g. mitotic cells detach easily from slide during fixation and staining procedures) and good penetration of PI-Rnase to allow full degradation of the RNA which otherwise will obscure the DNA-histogram.

(Selective) cell loss:

If cells detach too easily from the slide, try acid-washing and/or coating slides before seeding.

Acid wash:

• Wash 1h-o.n. in concentrated HCl
• Rinse well with ddH2O (10x)
  when coating coverslips it helps to boil in ddH2O after several washings for 15 min and rinse again 5x.
• Rinse well in 95% ETOH
• Airdry
• Store dry and sterile

Polylysine coating:

• Works best on acid-wash slides
• Incubate 10-15 min with polylysine (30-70.000 kd), 100 mg/ml
  (stock of 20 mg/ml can be stored at –20’C)
• Rinse 3x well in sterile H2O (polylysine in solution is toxic)
• Seed cells