TBARS ASSAY FOR LIPID OXIDATION


We will “cook” and test our harvested cell pellets for malondialdehyde (MDA), a byproduct of lipid oxidation. This will help us verify or disprove our hypothesis that antioxidant treatment can reduce lipid oxidation in cultured meat.

Water
TCA assay reagent
Color Reagent
MDA stock (125uM)
96-well plates
Ice

Sample cooking

  1. Place pellet tubes in a 100C heat block for 10 minutes. Retrieve samples from the heat block.

Standard preparation

  1. Prepare your standard curve:

Tube

MDA (uL)

Water (uL)

MDA concentration (uM)

A

0

500

0

B

20

480

5

C

40

460

10

D

100

400

25

E

200

300

50

Sample preparation

  1. Add 200 uL water to each cell pellet and mix
  2. Lyse cells using freeze-thaw method.
    1. Place tube in –80C for 5 minutes
    2. Completely thaw
    3. Repeat for 3 cycles

Assay setup

  1. Add 100 uL of sample or standard to appropriately labeled 1.5 mL Eppendorf tube (11 tubes total: 5 standards, 6 samples)
  2. Add 100 uL of TCA assay reagent and pipette to mix
  3. Add 800 uL of Color reagent to tubes
  4. Cap vials and place on 100C heat block for 1 hr
  5. Set microcentrifuge to 4C
  6. After 1 hour, remove vials and place on ice for 10 minutes
  7. After 10 minutes, centrifuge vials for 10 minutes at 1,600 x g and 4C
    1. Vials are stable at RT for 30 minutes
  8. Load 200 uL (in triplicate) from each vial into a clear 96-well plate. Avoid bubbles and keep track of where in the plate you have placed which standards / samples.
  9. Read absorbance at 535 nm

Assay analysis

  1. Prepare a scatter plot of absorbance/MDA control (uM) and add a linear best-fit line
  2. Using the y-intercept and slope of the best-fit line, calculate MDA uM from each of your samples, average between replicates and compare between conditions
  3. Hint: You will be plotting the standard curve alone. Your experimental samples will be plugged into the equation you got from the curve and presented in a separate graph.

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








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