View source for MTT Assay - Steps

==Title:==
MTT Assay - Steps

==Researches:==

1. Remove the culture medium from cell monolayers cultured in 96-well plates. 
2. Reconstitute each well with 0.2 mL supplemented RPMI-1640 containing 1 mg/mL MTT.
 3. Incubate the cultures for 2–4 hours at 37°C. 
4. Remove the supernatants from the wells.
 5. Add 0.2 mL per well of a lysing reagent containing 0.04 N HCl in isopropanol.
 6. Mix the content of the wells thoroughly. 
7. Read the plates in an automated microplate spectrophotometer at 570 and 630 nm as reference.

References 
Mossman, T. (1983). Rapid colorimetric assay for cellular growth and survival. Journal of Immunological Methods, 65 (1-2), 55-63. Sieuweters, A. M., Klijn, J. G., Peters, H. A., & Foekens, J. A. (1995; Nov. 33). MTT tetrazolium salt assay scrutinized. Eur J Clin Chem Clin Biochem, 11, 813-823. Darzynkiewicz, Z., Li, X., & Gong, J. (1994). Assays of cell viability: discrimination of cells dying by apoptosis, Methods in Cell Biology, 41, 15-38.



Statistical Analysis 
Null Hypothesis: There is no significant difference in the mean value between two groups, i.e. µ1 = µ2 
Alternate Hypothesis: There is a significant difference in the mean value between two groups, i.e. µ1 ≠ µ2 Level of Significance: α = 0.05 Statistical test used: t test Decision: The p value and the level of significance are compared. If p < 0.05, the null hypothesis is rejected and the alternate hypothesis is accepted. If p ≥ 0.05, the null hypothesis is accepted. 
Computations: The tables below give us the various computations and the p value. Comparison of the change in MTT Assay at Day 0 and Day 20 in study group: In study group, there was an increase in mean MTT Assay from Day 0 to Day 20. The increase in MTT Assay from Day 0 to Day 20 in Study Group was found to be statistically significant (p < 0.001).

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Summary
♦ The MTT assay is a well-established protocol for the study of cell viability and metabolism (Mitochondria). 
♦ There was a dramatic increase in mitochondrial activity in the study group. 
♦ Normally, processes like yoga or exercise or meditation increase mitochondrial activity by about 40-50%. 
♦ This study conclusively showed approximately 1000% increase of mitochondrial activity (cell viability) in the IA participants. 

Conclusion 

♦ The effective transmission of healing energy to participants by an adept healer through the process of energy darshan leads to Kundalini awakening to a statistically significant degree. 
♦ The addition of yoga and meditation along with energy darshan leads to greater increase in cellular energy.

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[[Category: Researches]]