1. Seed 200 µl of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit
are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µl of
10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2. Incubate the cells for overnight at 37°C, 5% CO2.
3. Treat the cells as desired.
4. Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5. Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is
used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. During the
incubation, the plates should be sealed with Parafilm.
Note: Fixing Solution is volatile. Wear appropriate personal protection equipment when using this chemical.
6. Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for five minutes each time with
gente shaking on the orbital shaker. The plate can be stored at 4°C for a week. Note: For all wash steps, tap the plate
gently on absorbent papers to remove the solution completely.
7. Add 100 µl Quenching Buffer and incubate for 20 minutes at room temperature.
8. Wash the plate 3 times with 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
9. Add 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
11. Add 50 µl of 1x Primary Antibodies or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and
incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room
temperature with gentle shaking on the shaker.
12. Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
Add 50 µl of 1x secondary antibodies (HRP-Conjugated Anti- Rabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG
Antibody) to corresponding wells and incubate for 1.5 hours at room temperature with gentle shaking on the shaker.
Note: Add HRP-Conjugated Anti-Rabbit IgG Antibody to the wells incubated with Primary Antibodies (rabbit, polyclonal)
and add HRP-Conjugated Anti-Mouse IgG Antibody to the wells incubated with Anti-GAPDH Antibody (mouse, monoclonal).
14. Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.
15. Add 50 µl of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark with
gentle shaking on the shaker.
Note: Ready-to-Use Substrate is a light-sensitive reagent. Keep away from light.
16. Add 50 µl of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.
Crystal Violet binds to cell nuclei and gives absorbance eadings proportional to cell counts at 595 nm.
17. After finishing reading the absorbance at 450 nm, wash the plate twice with 200 µl of Wash Buffer and twice with
200 µl of 1x TBS for 5 minutes each. Tap the plates on paper towel to remove the excess liquid. Let plate air dry for 5 mins
at room temperature.
18. Add 50 µl of Crystal Violet Solution to each well, incubate for 30 minutes at room temperature on the shaker.
Note:Crystal Violet is an intense stain. Avoid contact with skin and clothing.
19. Tip off Crystal Violet solution into beaker. Wash plate by dipping into bucket of water in the sink with the water
continuing to run. Carefully rinse the wells in ddH2O until no more color comes off the wells. Allow the plate to dry for
20. Add 100 µl of SDS Solution into each well and incubate on the shaker at room temperature for 1 hour.
21. Read absorbance at 595 nm with microplate reader. If absorbance is too high, the solubilized Crystal Violet Solution
can be diluted 10 times with ddH2O on a separate 96-well plate.