Hi Sivan,

You can stain cells in solution with dapi (1:10000) directly, then wash. to get them on a slide you can do a cytospin or resuspend in low volume and do the same thing you do with a blood smear. In both cases you let airdry or fix in 100% cold acetone. I would stain with dapi after they are on a slide.

Nate

Dear Sivan Schreier, do you want to stain them alive -as for viability- or have them fixed? To get them stuck on a surface you can use poly-L-lysine. Coat a coverslip under sterile conditions, and put it into the well, then you can put the non-adherent cells on top. With time and decantation some of them will eventually get stuck onto the coverslip.

Best,

Daniel

Simple flame fixation can also work, just let the dry overnigt before.

Hello

You can use the following protocol:

1-Collect a cell suspension of 2 × 105 to 1 × 106 cells.

2-Pellet the cells by centrifugation and discard the supernatant.

3-Tap the tube to resuspend the pellet in the residual liquid and add 1 mL of PBS at room temperature.

4-Transfer the full volume of resuspended cells to 4 mL of absolute ethanol at –20°C by pipetting the cell suspension slowly into the ethanol while vortexing at top speed.Leave the cells in ethanol at –20°C for 5–15 minutes.

5-Pellet the cells by centrifugation and discard the ethanol.

6-Tap the tube to loosen the pellet and add 5 mL of PBS at room temperature. Allow the cells to rehydrate for 15 minutes.

Counterstaining protocol :

1-Dilute the DAPI stock solution to 3 µM in staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P-40). A 1 mL volume will be required for each cell sample.

2-Centrifuge the cell suspension (from step 2.6) and discard the supernatant. Tap to loosen the pellet and add 1 mL of DAPI diluted in staining buffer.

3-Incubate for 15 minutes at room temperature.

4-Analyze by flow cytometry in the presence of the dye. If the cells are to be viewed by fluorescence microscopy, centrifuge the sample, remove the supernatant and resuspend cells in fresh buffer. Apply a drop of the suspension to a microscope slide, cover with a coverslip and view.

Good Luck