Mitochondrial Assay—The Study

Title:

Mitochondrial Assay—The Study

Researches:

a. 10 random blood samples from Inner Awakening® participants from above 50 years of age as study group b. Blood samples were taken on Day 0 and Day 18 c. Labeling was double blinded and sent to the laboratory for MTT assay d. All participants received the powerful ‘energy darshan’ (shaktipat) from Paramahamsa Nithyananda every day, in addition to participating in yoga, meditation and cathartic processes.

Fig: ROS mediated cell damage Since the etiology of many diseases is currently being quoted as related to free-radical mediated damage, mitochondria seemed to be the right subject for study and that is where we started. Genetics also plays a major role in the etio-pathogenesis of diseases. Genetic studies were carried out on participants who had significant health recoveries. QEEG studies were also done to study the effect on brain waves.

Laboratory Analysis The MTT assays were performed at Post Graduate Department of Biotechnology, Mangalore University. All results including the physical, physiological measurements and the MTT assay were statistically evaluated by professional biostatistician Dr. K. P. Suresh, Scientist (Bio Statistics), National Institute of Animal Nutrition and Physiology, Bangalore. Material and Methods This colorimetric assay is dependent on the ability of viable cells to metabolize a water-soluble tertazolium salt (yellow color) into a water insoluble formazan product (purple color). It measures the activity of cellular respiratory enzymes that reduce MTT. Since only viable cells perform this activity, the assay is used to measure the viability and proliferation of cells. Procedure: Isolation of Mononuclear Cells (MNC) Human Mononuclear cells can be separated from heparinized blood samples or from the buffy coat of centrifuged blood

sample with sodium citrate as an anticoagulant after removal of the erythrocytes and the plasma. The residual fraction (25–40 ml) contains more than 90% of the leukocytes, 10% of the erythrocytes, and 5% of the plasma.

1. Dilute buffy coats 1:4 (v/v) or heparinized blood samples 1:2 (v/v) with Dulbecco’s Phosphate buffered saline (PBS) without Ca2+ and Mg2+. 2. To a 10 ml centrifuge tube add 2 ml of defibrinated or anticoagulant treated blood and an equal volume of balanced salt solution (final volume 4 ml). 3. Mix the blood and buffer by inverting the tube several times or by drawing the mixture in and out of a pipette.

Procedure for Isolation of Mononuclear Cells

1. Invert the Ficoll-Paque media bottle several times to ensure thorough mixing. 2. Using aseptic techniques, add Ficoll-Paque media (3 ml) to the centrifuge tube. 3. Carefully layer the diluted blood sample (4 ml) onto the Ficoll-Paque media solution. 4. Centrifuge at 400 g for 30 to 40 minutes at 18ºC to 20°C (brake should be turned off). 5. Draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the mononuclear cell layer undisturbed at the interface. 6. Transfer the layer of mononuclear cells to a sterile centrifuge tube using a sterile pipette.

Washing the Cell Isolate

1. Estimate the volume of the transferred mononuclear cells. Add at least 3 volumes (~ 6 ml) of balanced salt solution to the mononuclear cells in the centrifuge tube. 2. Suspend the cells by gently drawing them in and out of a pipette. 3. Centrifuge at 400 to 500 × g for 10 to 15 minutes at 18°C to 20°C.

A Micro-titer Plate after MTT Assay Tetrazolium salts are converted by metabolically active cells to formazan, which can be quantified by measuring absorbance at 570 nm.

4. Remove the supernatant. 5. Re-suspend the mononuclear cells in 6 to 8 ml balanced salt solution. 6. Centrifuge at 400 to 500 × g (or 60 to 100 × g for removal of platelets) for 10 min at 18°C to 20°C. 7. Remove the supernatant. 8. For cell counts, dilute first 1:10 in supplemented RPMI -1640, and then 1:2 with 0.1% trypan blue solution, and count the cells with a Hemocytometer.