- Phosphate Buffered Saline:-
To 800 ml of distilled water, add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4. Adjust the pH to 7.4 with HCl. Add distilled water to a total volume of 1 liter. Dispense the solution into aliquots and sterilize them by autoclaving (20 min, 121°C, liquid cycle). Store at room temperature.
- 2% Para formaldehyde
- 0.2% BSA and 0.75% saponin
- Fluorescent Phallotoxin
- Pellet out 50,000 cells onto each cover slip placed inside a 12- well plate.
- Incubate the cells for 24 hours at 37˚C in a carbon dioxide incubator.
- Take it out and check for 90% confluency under a phase contrast microscope.
- Perform scratch assay.
- Remove all the media from the wells using a pipette tip attached to a vacuum pump.
- Wash two times with PBS.
- Fix the cells with (500 microlitre) 2% Para formaldehyde supplemented with 1mM Magnesium chloride and 0.1mM Calcium chloride.
- Incubate the culture plate for 30 minutes in the dark.
- Wash again with PBS for three times thoroughly.
- Block with 0.2% BSA and 0.75% saponin and incubate again in the dark for 15 minutes.
- Incubate for 20 minutes with PBA and phalloidin in the ratio of 1:200 . To avoid evaporation, keep the well plate inside a covered container during the incubation.
- Wash three times with PBS.
- Take the cover slips out of the wells using a forceps. Care should be taken to avoid breakage of the cover slip. Invert it and mount on slides.
- Fix the cove rslip onto the slide by using nail polish remover. Use a forceps and gently press the middle of the coverslip and use the remover around it touching both the coverslip and slide.
- View it under fluorescence at 20X.
Cite this Simulator: