https://bridgeslab.sph.umich.edu/protocols/api.php?action=feedcontributions&feedformat=atom&user=Sotu 2026-06-02T02:19:02Z User contributions MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=Serum_Total_Cholesterol_Assay&diff=2691 2024-01-18T17:33:31Z

<p>Sotu: </p> <hr /> <div>== Materials ==<br /> * Serum, need at most 10 uL per mouse, at little as 2 uL per mouse. See [[Collecting_and_Storing_Mouse_Serum]]<br /> * Infinity cholesterol reagent (Thermo Cat# TR13421)<br /> * Cholesterol standard (Pointe Scientific). Also make a 10x dilution.<br /> * Clear 96 well plate (we use Thermo Cat# 130188)<br /> * Plate reader<br /> <br /> == Protocol ==<br /> * Draw out plate including samples, plan for 2-3 replicates for each sample<br /> ** 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<br /> * Add 100 uL cholesterol reagent to each well using the multichannel pipet.<br /> * Start a timer <br /> * Add 2 uL serum (mouse sample) to each well<br /> * 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)<br /> * 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.<br /> * Once all samples are added, incubate plate at room temperature for 15 minutes<br /> <br /> === Plate Reader ===<br /> *Turn on computer, screen, and plate reader (switch on back right)<br /> *Log in<br /> *Open SoftMaxPro 5.3<br /> *Measure absorbance at 500 nm (Under settings, set Lm1 to 490. Our machine does not have a filter for 500 nm)<br /> *Click Read<br /> *File —&gt; Print<br /> *Save —&gt; FileName YYYY-MM-DD-Chol <br /> *Save in Bridges Folder<br /> *Export —&gt; samename.txt<br /> *Get txt file via USB drive and print<br /> <br /> <br /> == Calculations ==<br /> * Draw a standard curve of Cholesterol amount (in ug) versus absorbance<br /> * From the slope of that curve (or a linear model) calculate the amount of cholesterol per well, divide that by two<br /> * Average the replicates. Repeat if technical variation is too high</div>

Sotu https://bridgeslab.sph.umich.edu/protocols/index.php?title=Serum_Total_Cholesterol_Assay&diff=2690 2024-01-18T17:32:04Z

<p>Sotu: added directions for plate reader</p> <hr /> <div>== Materials ==<br /> * Serum, need at most 10 uL per mouse, at little as 2 uL per mouse. See [[Collecting_and_Storing_Mouse_Serum]]<br /> * Infinity cholesterol reagent (Thermo Cat# TR13421)<br /> * Cholesterol standard (Pointe Scientific). Also make a 10x dilution.<br /> * Clear 96 well plate (we use Thermo Cat# 130188)<br /> * Plate reader<br /> <br /> == Protocol ==<br /> * Draw out plate including samples, plan for 2-3 replicates for each sample<br /> ** 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<br /> * Add 100 uL cholesterol reagent to each well using the multichannel pipet.<br /> * Start a timer <br /> * Add 2 uL serum (mouse sample) to each well<br /> * 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)<br /> * 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.<br /> * Once all samples are added, incubate plate at room temperature for 15 minutes<br /> <br /> === Plate Reader ===<br /> *Turn on computer, screen, and plate reader (switch on back right)<br /> *Log in<br /> *Open SoftMaxPro 5.3<br /> *Measure absorbance at 500 nm (Under settings, set Lm1 to 490. Our machine does not have a filter for 500 nm)<br /> *Click Read<br /> *File —&gt; Print<br /> *Save —&gt; FileName YYYY-MM-DD-Chol <br /> *Save in Bridges Folder<br /> *Export —&gt; samename.txt<br /> *Get txt file via USB drive and print<br /> <br /> <br /> == Calculations ==<br /> * Draw a standard curve of Cholesterol amount (in ug) versus absorbance<br /> * From the slope of that curve (or a linear model) calculate the amount of cholesterol per well, divide that by two<br /> * Average the replicates. Repeat if technical variation is too high</div>

Sotu https://bridgeslab.sph.umich.edu/protocols/index.php?title=Serum_Total_Cholesterol_Assay&diff=2689 2024-01-18T16:44:28Z

<p>Sotu: added clarifying notes</p> <hr /> <div>== Materials ==<br /> * Serum, need at most 10 uL per mouse, at little as 2 uL per mouse. See [[Collecting_and_Storing_Mouse_Serum]]<br /> * Infinity cholesterol reagent (Thermo Cat# TR13421)<br /> * Cholesterol standard (Pointe Scientific). Also make a 10x dilution.<br /> * Clear 96 well plate (we use Thermo Cat# 130188)<br /> * Plate reader<br /> <br /> == Protocol ==<br /> * Draw out plate including samples, plan for 2-3 replicates for each sample<br /> ** 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<br /> * Add 100 uL cholesterol reagent to each well using the multichannel pipet.<br /> * Start a timer <br /> * Add 2 uL serum (mouse sample) to each well<br /> * 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)<br /> * 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.<br /> * Once all samples are added, incubate plate at room temperature for 30 minutes<br /> * Measure absorbance at 500 nm<br /> <br /> == Calculations ==<br /> * Draw a standard curve of Cholesterol amount (in ug) versus absorbance<br /> * From the slope of that curve (or a linear model) calculate the amount of cholesterol per well, divide that by two<br /> * Average the replicates. Repeat if technical variation is too high</div>

Sotu https://bridgeslab.sph.umich.edu/protocols/index.php?title=User:Sotu&diff=2683 2023-10-28T13:50:47Z

<p>Sotu: /* Cholesterol Assay */</p> <hr /> <div></div>

Sotu https://bridgeslab.sph.umich.edu/protocols/index.php?title=User:Sotu&diff=2682 2023-10-28T13:48:46Z

<p>Sotu: Created page with &quot;== Cholesterol Assay == === Materials === * Reagent * Cholesterol Standard === Protocol === * Make 10X stock dilution: Add 50ɥL Cholesterol Standard 450ɥL water to a 1.5mL...&quot;</p> <hr /> <div>== Cholesterol Assay ==<br /> === Materials ===<br /> * Reagent<br /> * Cholesterol Standard <br /> === Protocol ===<br /> * Make 10X stock dilution: Add 50ɥL Cholesterol Standard 450ɥL water to a 1.5mL tube<br /> * Pipette 100ɥL of Reagent to 22 wells<br /> * Add 2-10ɥL of 10X stock dilution to wells in columns 2-6 in increments of 2ɥL. Each column will have 2 replicates per volume (1 replicate per row). <br /> * Repeat in columns 7-11 with 2-10ɥL of Cholesterol Standard (1X dilution) <br /> * Allow reaction to proceed for 15 minutes<br /> * Read absorbance</div>

Sotu