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Actin Assembly
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 Materials Required

 

  1. 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. 2% Para formaldehyde
  3. 0.2% BSA and 0.75% saponin
  4. Fluorescent Phallotoxin

 

Procedure

 

  1. Pellet out 50,000 cells onto each cover slip placed inside a 12- well plate.
  2. Incubate the cells for 24 hours at 37˚C in a carbon dioxide incubator.
  3. Take it out and check for 90% confluency under a phase contrast microscope.
  4. Perform scratch assay.
  5. Remove all the media from the wells using a pipette tip attached to a vacuum pump.
  6. Wash two times with PBS.
  7. Fix the cells with (500 microlitre) 2% Para formaldehyde supplemented with 1mM Magnesium chloride and 0.1mM Calcium chloride.
  8. Incubate the culture plate for 30 minutes in the dark.
  9. Wash again with PBS for three times thoroughly.
  10. Block with 0.2% BSA and 0.75% saponin and incubate again in the dark for 15 minutes.
  11. 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.
  12. Wash three times with PBS.
  13. 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.
  14. 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.
  15. View it under fluorescence at 20X.

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