CELL VIABILITY OF ANTIOXIDANT TREATED MUSCLE CELLS

General Notes

PrestoBlue is light sensitive so make sure to keep protected from light whenever possiblePrestoBlue measure cell metabolism as a proxy for cell number (and general cell health)
PrestoBlue is light sensitive so make sure to keep protected from light whenever possible
PrestoBlue measure cell metabolism as a proxy for cell number (and general cell health)

Materials

Antioxidant treated BSCs
PrestoBlue reagent
96-well plate
Plate reader
BSC GM
DMSO
Acetone
Beta carotene
Carrot juice
Lycopene
V8 juice
Curcumin
Turmeric
Filters150 mL bottle top filters for the juices Syringe filters and syringes for the powders
150 mL bottle top filters for the juices
Syringe filters and syringes for the powders
Conical Tubes
T175 Flasks
BSC cell suspension

Method

Viability Test

1. Add 20 µL PrestoBlue to each well
2. Add controls: to 3 empty wells, add 200 µL BSC-GM and 20 µL PrestoBlue to each well (this will be your “blank” reading)
3. Tilt plate to ensure PrestoBlue reagent is spread across well
4. Cover plate in foil and return to incubator
5. Incubate ~1 hour
6. Transfer 150 µL media + PrestoBlue from each experimental well into a 96 well plate (note which sample is in which well, see schematic below for reference)
7. Read plate at 560/590 nm
8. Analyze results:
   1. Subtract control/blank readings from each sample (average the blank readings and subtract this number from each experimental sample’s reading)
   2. Create a bar chart comparing each treatment
   3. Note any differences in cell viability with each treatment

Media Prep

1. Set aside 80 ml of BSC GM (this is the control group)
   1. Can keep this in your original bottle, but ensure there’s 80mL left
2. Prepare 80 ml of growth media with your chosen concentration of carotenoid (pure extract)
3. Prepare 80 ml of growth media with your chosen concentration of carotenoid (from juice)

Cell Seeding

1. Take a tube of 9mL cell suspension and divide evenly into 3x15 ml conical tubes (~3mL each)
   1. BE SURE to evenly mix/homogenize the tubes prior to dividing them amongst the tubes
2. Label the tubes according to the 3 media your prepared
3. Centrifuge your tubes at 300 x g for 5 mins
4. Remove the supernatant from the spun down cell suspension
5. Add 5 ml of each media into its appropriate tube with cells and mix/homogenize the solution well
   1. Ensure that the solution is homogenous/single cell suspension/free of cell clumps
6. Add remaining 20 mL of each media to separate T175 flasks
   1. Label with the sample group, your group #, the class # and section, and the date
7. Add each 5 ml of cells + carotenoid media/normal media into each appropriate flask.
8. Transfer the flasks to 37C 5% CO2.
9. Feed on Sun and Tues.

Reference volumes:

Vessel	Surface (cm2)	~PBS volume	~Trypsin volume	~Media volume
6-well plate	9.6	1 mL	500 uL	2 mL
12-well plate	3.5	500 uL	250 uL	1 mL
24-well plate	1.9	500 uL	250 uL	1 mL
48-well plate	1.1	200 uL	100 uL	500 uL
96-well plate	0.32	100 uL	50 uL	200 uL
T-25	25	3 mL	1 mL	5 mL
T-75	75	5 mL	2 mL	12 mL
T-175	175	10 mL	3 mL	30 mL

References

1. https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0018371-PrestoBlueHS-CellViabilityReagent-PI.pdf

This protocol was developed for BME 174: cultivated meat laboratory course at Tufts University. The course is administered by Professor David Kaplan's laboratory.

PROTOCOL DEX