Flash link: https://vlab.amrita.edu/repo/BIOTECH/CEL/Cell_Attachment/index.swf

Materials Required

1. Gelatin 2mg/ml in 10mM Acetic acid.
2. Bovine serum albumin (1% in PBS) heat inactivated at 80 degree Celsius for  3 minutes.
3. 96- well plate.
4. Glass slide.
5. Cover slip.
6. Hemocytometer.
7. 50 ml centrifuge tubes.
8. Crystal violet solution.
9. Cells.
10. Trypsin -EDTA.
11. Phosphate buffered saline (PBS).

 Procedure

1. Coat the wells of the 96-well plate with serially diluted gelatin (2mg/ml to 62.5%).
2. Transfer 100µl of  the PBS solution into each  well.
3. Add 100µl of gelatin into the wells of the plate containing 100µl of PBS.
4. Add 100µl of BSA to the microtiter plate. Repeat the same step 4,5,6 for plate well “B”also.
5. Now Add 100 ul of media to all the wells.
6. Add 100 µl of 10% serum to the first well of C and mix gently. Then take 100ul of the solution from the same well and add it to the second well till the 10th well.
7. Incubate the plate for 1 hr in the incubator.
8. Wash the excess with 50 ul of PBS   5 times .
9. Block with 100 µl of BSA  for 30-45 min at room temperature (25 degree C).
10. Wash 5 times with PBS.
11. Now take out the petriplates containing the cells  from the  CO₂ incubator.
12. Trypsinize  the cells from 10 cm Petri dish (tissue culture grade). Suction out media gently using a 200µl pipette tip.
13. Gently add PBS  twice to the corner of the plate without disturbing the cell layer.
14. Remove PBS from the plate.
15. Pipette trypsin- EDTA  onto the washed cell monolayer using 1ml per 25cm² of surface area. Rotate the plate to cover the monolayer with trypsin. Decant the excess trypsin.
16. Incubate for 5 min at 370C.
17. After 5 mins, take the plate back to the LAF and then tap it.
18. Resuspend the cells in a small volume of fresh serum-containing media to inactivate the trypsin.
19. Check under inverted  microscope time to time to verify the degree of dissociation. Tap gently to shake off the cells from surface.
20. Transfer the cell suspension to a 15ml tube.
21. Centrifuge the cell suspension at 500rpm for 5 minutes.
22. Carefully remove the supernatant without disturbing the cell pellet.
23. Resuspend to minimal volume (10mL) of serum free media.
24. Count the cells to 4x105cells/mL using a hemocytometer.
25. Add 100µl of cell suspension at each well (40,000 cells per well) and Incubate for 2 hr.
26. Wash the non-adhered cells using distilled water (50µl).
27. Fix the cells in 50ul of fixative (formalin ) for 30 min.
28. Wash off with 100 µl water. Shake off gently and  air dry for 15 minutes.
29. Add crystal violet solution (50µl) for 15 min.
30. Wash off with water (100µl) 3 times and air dry.
31. Add 10% acetic acid 100 to all the wells and measure the OD at 590 nm.