How to Determine Cytotoxic Activity of Crude extracts by MTT assay

-
After total flavonoids content (TFC), total polyphenolics content (TPC), and antioxidant activity of different crude extracts of orchid including Dondrobium longicornu have been estimated, we decided to determine their cytotoxic activity using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on human brain tumor cells (U251) and cervical cancer cells (HeLa).  We employed previously established protocols (Mosmann, 1983; Sargent and Taylor, 1989) which is presented below point wisely. 

  1. Culture human brain tumor cells (U251) and cervical cancer cells (HeLa) in RPMI 1640 medium and incubate them under 5% CO2 at 37° C for 48 hours to reach  80% confluence.
  2. Harvest by gentle scraping with a cell scraper and resuspended in the medium. 
  3. Dispense 100 μL of such medium with 5 × 103 cells into each well of 96-well microtiter cell culture plate and incubated under the same conditions of cell culture for 48 hours to allow adherence and growth of cells.
  4. Gently aspirate supernatants and added 100μL of Dendrobium longicornu extracts over a range of four cytotoxic concentrations (100 – 800   μg/mL) prepared in medium and incubated for 24 hours.
  5. Dissolve formazan crystals which are formed after addition of extract addition in 100 μL DMSO. 
  6. Read the plate on a microplate reader (iMarkTM, Bio-Rad) at 595 nm. 
  7. Calculate the number of dead cells per well as a percentage of the control; thus, measuring cell death after drug exposure.  
  8. Plot dose-response curve for each extract to calculate the inhibition of cell growth by 50%. 
  9. Finally, calculate IC50 of extracts from the regression equation where the abscissa represents the series of the concentration of extracts and ordinate the triplicate percentage inhibition of cell growth. 

For further details, you could visit the link given below:
Paudel, M. R., Chand, M. B., Pant, B., & Pant, B. (2017). Cytotoxic Activity of Antioxidant-Riched Dendrobium longicornu. Pharmacognosy Journal, 9(4), 499–503. https://doi.org/10.5530/pj.2017.4.81

 References:
1. Mosmann T. ( 1983). Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. Journal of Immunological Methods 65(1-
2):55-63. doi:10.1016/0022-1759(83)90303-4. https://doi.org/10.1016/0022-
1759(83)90303-4.

 2. Sargent JM, Taylor CG. (1989). Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. British Journal of Cancer 60(2):206-10. https://doi.org/10.1038/bjc.1989.252. 

Comments

Post a Comment

Popular posts from this blog

How we performed the estimation of total flavonoid content ?

-
Image
This time I am sharing a method for the estimation of total flavonoid content in our experiments. We Flowchart. the process of estimating total flavonoids content. followed a previously done standard protocol  (Quettier  et al.  2000 and Stankovic 2011)  with slight modification.  Firstly, we  mixed  e ach plant extract (2 ml; 0.5 mg/ml) separately  with aluminium chloride solution (1 ml; 2 %). Then, we allowed the reaction mixture to stand for an hour at room temperature. Again, we measured the absorbance of the mixture at 415 nm using the UV-spectrophotometer. We  obtained  calibration curve (see Fig 1.) using quercetin solution series (25, 50 75, and 100 µg/ml) as a standard. Here, we used the reagent mixture with the same volume of ethanol instead of plant extract solution as a blank. Thus, the obtained curve was calibrated to estimate the concentration of total flavonoid in each plant extract. The total ...
Read more

How to Align Fastq Files of Sample Genomic DNA Sequence to Reference Genome

-
Image
Since the aim of this blog is to share the information on various aspects of plants and plant sciences, this time, I am blogging about the bioinformatics tools that help study about the genome of organisms including plants.    The application of  BWA  software or any other software that allows alignment of fastq (.fq) files of the sample genomic DNA sequence to  reference genome  is an essential step before carrying out further investigations such as the genome analysis. In this post, I am going to present the instruction for the alignment of quality trimmed fastq (.fq) files of a sample genome to the reference genome using BWA (Burrows-Wheeler Aligner) software. This instruction covers installing of BWA software, indexing the reference genome, quality trimming the raw fastq files, and aligning the quality trimmed  fastq  files to reference genome to get  SAM  (Sequence Alignment Map) file of a sample genome.  Image 1. The vi...
Read more

The Rapid Method of Estimating Antioxidant Activity and Total Polyphenols Content of Sample Plant Extracts

-
Image
Estimation of total polyphenols content Figure1. The appearance of bluish-black colour after the addition of reagents on sample plant extracts during the estimation of total polyphenols content.  We estimated  total polyphenol content  by a standard protocol (Singleton et al . 1999 and Stankovic 2011) with slight modification. Briefly, we  separately mixed  each plant extract (0.5 ml; 1 mg/ml)  with the Folin-Ciocalteu phenol reagent (2.5 ml; 10%) and aqueous sodium bicarbonate solution (2.5 ml; 7.5%). Then, the reaction mixture was allowed to stand for about 45 minutes and the absorbance was measured at 765 nm using the UV- spectrophotometer (Thermo Fisher Scientific, Genesystem -10.5).The calibration curve was plotted using gallic acid as standard in ethanol (absolute) using the concentration series of 25, 50, 75, and 100 µg/ml. Here, the reagent mixture with the  same volume of ethanol instead of plant extract solution was used ...
Read more