Serum Total Cholesterol Assay - Revision history

Sotu at 17:33, 18 January 2024

2024-01-18T17:33:31Z

← Older revision		Revision as of 17:33, 18 January 2024
Line 16:		Line 16:
	* Once all samples are added, incubate plate at room temperature for 15 minutes		* Once all samples are added, incubate plate at room temperature for 15 minutes

	=== Plate Reader ===		=== Plate Reader ===
	*Turn on computer, screen, and plate reader (switch on back right)		*Turn on computer, screen, and plate reader (switch on back right)
	*Log in		*Log in

Sotu: added directions for plate reader

2024-01-18T17:32:04Z

added directions for plate reader

← Older revision		Revision as of 17:32, 18 January 2024
Line 14:		Line 14:
	* 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)		* 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.		* 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 30 minutes		* Once all samples are added, incubate plate at room temperature for 15 minutes
	* Measure absorbance at 500 nm
			=== 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 ==		== Calculations ==

Sotu: added clarifying notes

2024-01-18T16:44:28Z

added clarifying notes

← Older revision		Revision as of 16:44, 18 January 2024
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	* Add 100 uL cholesterol reagent to each well using the multichannel pipet.		* Add 100 uL cholesterol reagent to each well using the multichannel pipet.
	* Start a timer		* Start a timer
	* Add 2 uL serum to each well		* Add 2 uL serum (mouse sample) to each well
	* In the middle of the time add the standards		* 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.		* 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 30 minutes		* Once all samples are added, incubate plate at room temperature for 30 minutes

Davebrid: Created intitial protocol

2024-01-18T16:06:47Z

Created intitial protocol

New page

== 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 to each well
* In the middle of the time add the standards
* 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 30 minutes
* Measure absorbance at 500 nm

== 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