A) SOFT AGAR METHOD. Principle: Soft agar method used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. Also to study cytotoxic nature of any new drug compound. Material Required: a. Reagent A: 2X plain media containing 20% F.B.S, 4mM L- Glutamine, 2X NEAA, 0.6% sodium bicarbonate, 2% sodium pyruvate, 200U/ml penicillin/Streptomycin. Composition for 100ml- 2X plain media (According to cell line): 73.4 ml FBS : 20 ml Glutamine(200mM) : 2 ml 2X NEAA (Non essential amino acids) : 0.6 ml Sodium pyruvate : 2 ml Antibiotics : 2 ml b. Reagent B: 1.2% Bacto Agar. (prewarm to 56 degree centigrade before use). c. Reagent C: 0.8% Bacto Agar. Protocol:
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A) SOFT AGAR METHOD.
Principle: Soft agar method used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. Also to study cytotoxic nature of any new drug compound.
b. Reagent B: 1.2% Bacto Agar. (prewarm to 56 degree centigrade before use).
c. Reagent C: 0.8% Bacto Agar.
Protocol:
1. A mixture of 50µl of Reagent A and 50µl of Reagent B were plated onto each well of a 96-well microtitre assay plate.
2. 10µl of cells (of specific cell count standardize for respective cell lines) were mixed with 20µl Reagent A and 30µl Reagent C + 2µl of drug, in a vial and transferred to the solidified prelayers of the assay plate.
3. Allow the cells to grow at 37 degree centigrade and 5% CO2 for 1 weeks.
4. When the cell colonies are developed score the colonies using Alamar Blue.
5. Measure the cell growth by reading the absorbance at 570nm.
Template when standardization of plating efficiency was done.In each pink block different cell density was plated.
Template when comparison with Cisplatin and CleI were did.
