NB-5 NB-6 Amsa 20 40 Compound Concentration (uM) 60 80 MIT Assay The cytotoxicit
ID: 123695 • Letter: N
Question
NB-5 NB-6 Amsa 20 40 Compound Concentration (uM) 60 80 MIT Assay The cytotoxicity of the compounds was tested against nine human tumor cell lines: HL-60 (acute promyelocytic leukemia). HL60/MX1 (mitoxantrone-resistant acute promyelocytic leuke- mia). CCRF-CEM (acute lymphoblastic leukemia), NG97 (glyo- blastoma) T47-D (mammary ductal adenocarcinoma), Raji Burkitt's lymphoma) and Jurkat (T-cell leukemia). All cell lines were obtained from Rio de Janeiro Tissue Cell Bank except NG97 cells that were kindly provided by University of São Paulo. Cells were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum, 2 mM glutamine, 100mg/mL streptomycin and 100 U/mL penicillin at 37 C with 5% CO2-For experiments, cells were plated in 96-well plates (10 cells/well. After 24h, the compounds (1 to 75 M) dissolved in DMSO were added to each well and incubated for 3 days (72h). Control groups received the same amount of DMSO (0.1%). Amsacrine was used as positive control. Growth of tumor cells was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)- 2.5-diphenyl-2H-tetrazolium bromide (MIT) to a blue formazan product. At the end of 72 h incubation, 20 Lof 0.5 mg/mL of MTT were added in each well, and cells were incubated again at 37 C with 5% CO-Three hours later, the formazan product of MTT reduction was dissolved in 20% SDS, and absorbance was measured using a multi-plate reader (EL808, Biotek, USA). Drug effect was quantified as the percentage of control absorbance of reduced dye at 570 nm.Explanation / Answer
Answer: Protocol for the above experiment is as follows:
Principle of the method: Growth of tumor cells is quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product. This enzymatic reaction can be utilised to quantify the tumor growth for a specific cell lines.
1. cell lines specific to particular cancer are taken and were cultured in RPMI-1640 medium, along with 10% fetal calf serum, 2mM glutamine,100 mg/mL streptomycin and100 U/mL penicillin at 37 degree celsius with 5% CO2.
2. For performing experiment the cells will be plated on 96 well plate (10^4 cells/well).
3. After 24 hours the compound (1 to 75 microM) dissolved in DMSO were incubated for 3 days i.e 72 hours. Controls groups also will receive same amount of DMSO (0.1%).
4. Amsacrine is used as a positive control were used in the plate.
5. At the end of 72 hours 20 microlitre of 0.5 mg/mL MTT is added in each well and cells were again incubated for 37 degree celsius with 5% CO2.
6. 3 hours later the formazan product of MTT reduction was dissolved in 20% SDS, and the absorbance was measured.
7. Drug effect quantification will be done as the percentage of control i.e Amsacrine absorbance of reduced dye at 570 nm.
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