1.0 INTRODUCTION:
The SRB assay is used for cell density determination, based on the measurement of cellular protein content. The sulforhodamine B (SRB) colorimetric assay is based on the ability of the SRB dye to bind basic amino acid residues on proteins.2.0 METHODOLOGY:
· Cell culture
· Mouse fibroblast L929 cell line will be procured from American Type Culture Collection USA, NCCS Pune, or from authorized vendors cultured from suitable media supplemented with FBS or FCS or as applicable.
· The cells will be maintained at 37°C under 5% CO2 and 100% humidity in DMEM and supplemented with 10% fetal calf serum and antibiotics (200 μl/ml penicillin G, 200 μg/ml streptomycin, and 2 μg/ml fungizone) and harvested during the logarithmic growth phase.
· After sufficient growth for experimentation, the cells will be trypsinized and plated in 96-cluster well culture plates at a concentration of 1 × 104 cells/well.
· Each well-contained 100μl of cell suspension and the plates will be incubated for 24 h at 37 °C under 5% CO2 to obtain a monolayer culture.
· After 24 h of incubation, the old medium will be removed from each well. Then, a 100-μl eluted volume from the test solution at different concentrations; the positive control; or negative control will be inserted into a 96-cluster well culture plate (8 wells/test material).
· Two 96-cluster well culture plates will be separately prepared to evaluate cell viability using SRB assays and experiments will be repeated in triplicate.
· Following a 24-h incubation period at 37 °C under 5% CO2, the cell viability of both plates will be assessed.
· Assay
· After an incubation period, cell monolayers will be fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min; then, excess dye will be removed by washing the cells repeatedly with 1% (vol/vol) acetic acid.
· The protein-bound dye will be dissolved in 10 mM Tris base solution for optical density (OD) determination at 510 nm using a microplate reader.
· Cell viability will be expressed as a percentage of the control values. The intra-class correlation coefficient (ICC) and limits of agreement statistics will be used to compare the scores. The limits of agreement statistics will also be used as a descriptive measure of agreement.
3.0 ENDPOINT PARAMETER(S):
· Cell viability
4.0 REFERENCE(S):
2.1 Vajrabhaya, Lo., Korsuwannawong, S. Cytotoxicity evaluation of a Thai herb using tetrazolium (MTT) and sulforhodamine B (SRB) assays. J Anal Sci Technol 9, 15 (2018). https://doi.org/10.1186/s40543-018-0146-0.
2.2 Esteban A. Orellana and Andrea L. Kasinski. Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation. Bio Protoc. 2016 Nov 5; 6(21): e1984. doi: 10.21769/BioProtoc.1984.
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