Serum Total Cholesterol Assay
Materials
- Serum, need at most 10 uL per mouse, at little as 2 uL per mouse. See Collecting_and_Storing_Mouse_Serum
- Infinity cholesterol reagent (Thermo Cat# TR13421)
- Cholesterol standard (Pointe Scientific). Also make a 10x dilution.
- Clear 96 well plate (we use Thermo Cat# 130188)
- Plate reader
Protocol
- Draw out plate including samples, plan for 2-3 replicates for each sample
- Include plans for standard curve, using 2, 4, 6, 8 uL of 10x diluted cholesterol standard, and 1, 1.5, 2 uL of the undiluted cholesterol standard
- Add 100 uL cholesterol reagent to each well using the multichannel pipet.
- Start a timer
- Add 2 uL serum (mouse sample) to each well
- In the middle of the time add the standards (2, 4, 6, 8 uL of 10x and 1, 1.5, 2 uL of the undiluted cholesterol standard)
- The assay takes 15 minutes to develop at room temperature, and is stable for 30 minutes. That means if the timer hits 30 minutes you wont be able to finish in time, so stop and start the plate.
- Once all samples are added, incubate plate at room temperature for 15 minutes
Plate Reader
- Turn on computer, screen, and plate reader (switch on back right)
- Log in
- Open SoftMaxPro 5.3
- Measure absorbance at 500 nm (Under settings, set Lm1 to 490. Our machine does not have a filter for 500 nm)
- Click Read
- File —> Print
- Save —> FileName YYYY-MM-DD-Chol
- Save in Bridges Folder
- Export —> samename.txt
- Get txt file via USB drive and print
Calculations
- Draw a standard curve of Cholesterol amount (in ug) versus absorbance
- From the slope of that curve (or a linear model) calculate the amount of cholesterol per well, divide that by two
- Average the replicates. Repeat if technical variation is too high