Sackler School of
Graduate Biomedical Sciences

Filipin staining of cultured cells

Reagents –
Dulbecco's PBS, pH 7.4 without calcium and magnesium

10% (w/v) Paraformaldehyde stock solution. Weigh 10 g PFA with mask and gloves. Add to 90 ml dw. Stir in chemical hood on hot plate. Bring to 60^oC with stirring. Add drops of 1N NaOH until the solution clarifies. Adjust pH to 7 using pH paper. Bring to 100 ml with dw and filter thru Whatman #1 paper. Store at 4^oC.

3% (w/v) paraformaldehyde (PFA) in PBS.
3 ml 10% PFA + 4.5 ml water + 2.5 ml 4X PBS.

Filipin (1 mg/40 uL of DMSO = 25 mg/ml). Store at -20^oC. Use within 2-3 weeks.

Method –
Cells are typically seeded and cultured in chamber slides (Falcon CultureSlide, BD Biosciences). Rinse the cells 3 times with PBS.

Fix cells with 3% PFA in PBS for 30 min at room temperature. Rinse 3x 5 min each with PBS on rotator. Quench PFA with 1.5 mg/ml glycine in PBS for 10 min at room temperature.

Stain with 25 ug/ml filipin in PBS for 30 min at room temperature. Rinse 3x 5 min each with PBS. Mount coverslips onto slides using 90% (v/v) glycerol in PBS.

View in fluorescence microscope using UV filter package for Hoechst stain.

Protocol kindly provided by Peter Pentchev, NINDS.
Reference: Jacobs et al. (1997)
Modified September 2006