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