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Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)

Reviews (10)Q&A (53)References (219)
HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)
  • Functional Studies - HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)
  • ab65390 HDL and LDL/VLDL Cholesterol Assay Kit
  • ab65390 HDL and LDL/VLDL Cholesterol Assay Kit

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric/Fluorometric
  • Platform: Microplate reader
  • Assay time: 1 hr
  • Sample type: Other biological fluids, Plasma, Serum, Tissue Lysate, Urine

Overview

  • Product name

    Cholesterol Assay Kit - HDL and LDL/VLDL
    See all HDL LDL/VLDL kits

  • Detection method

    Colorimetric/Fluorometric

  • Sample type

    Urine, Serum, Plasma, Other biological fluids, Tissue Lysate

  • Assay type

    Quantitative

  • Assay time

    1h 00m

  • Product overview

    Cholesterol Assay Kit ab65390 uses a simple method to quantify total cholesterol, free cholesterol, and cholesterol esters in mammalian samples. It also includes an easy method to separate HDL and LDL / VLDL cholesterol. 


    In the cholesterol assay protocol, cholesterol oxidase acts on free cholesterol to produce a chemical which reacts with a probe to generate color (570 nm) and fluorescence (Ex/Em = 538/587 nm). Cholesterol esterase is used to hydrolyze cholesteryl ester into free cholesterol.


    If cholesterol esterase is included in the assay, total cholesterol is measured. If it is not included, free cholesterol is measured. The amount of cholesterol ester can be calculated by subtracting free cholesterol from total cholesterol.


    Note: this kit is primarily designed for use with serum and plasma samples. It has been successfully used with liver samples (including in Chang et al. 2019, Yu et al. 2018, and Sadh et al. 2017); sample preparation in liver (and other tissue) samples may need optimization and modification by the user for successful use. 


    Cholesterol assay protocol summary:
    - use complete sample for cholesterol measurement; or to separate HDL and LDL/VLDL, mix sample with precipitation buffer, incubate for 10 min, centrifuge for 10 min at 2,000 g, keep HDL fraction supernatant, repeat centrifugation and resuspend LDL/VLDL fraction pellet
    - for assay, add samples and standards to wells
    - add total cholesterol reaction mix (with esterase) or free cholesterol reaction mix (without esterase) and incubate for 60 min at 37ºC
    - analyze with microplate reader

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K613 HDL and LDL/VLDL Quantification Colorimetric/Fluorometric Kit. K613-100 is the same size as the 100 test size of ab65390.

    Other cholesterol assay kits include:
    - Cell-based Cholesterol assay kit ab133116 
    - Cholesterol/Cholesterol Ester assay kit ab65359 
    - Cholesterol Efflux assay kit ab196985 
    - Cholesterol Uptake assay kit ab236212

    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

     

    How other researchers have used Cholesterol Assay Kit ab65390

    This cholesterol assay kit has been used in publications in a variety of sample types, including:
    - Human: serum1
    - Mouse: plasma and liver tissue2, plasma3, serum4,1
    - Rat: liver tissue5, serum1, plasma6
    - Fish: Z. temminckii serum7

    References: 1 - Lin C et al 2018; 2 - Change RC et al 2019; 3 - Hinder et al 2019, Havighorst A et al 2019, Guo X et al 2017, Wen et al 2018, Fernandes GW et al 2018, Ahnstedt H et al 2018, Dumas SN and Ntambi JM 2018; 4 - Liu J et al 2018, Cui XB et al 2019, Rohm et al 2018; 5 - Yu S et al 2018; 6 - Park JB 2017; 7 - Chae Y et al 2018

  • Platform

    Microplate reader

Properties

Images

  • HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)
    HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)Li G et al., PLoS One, 7, e35895, 2012 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Wild type (WT) or farnesoid X receptor knock-out (FRX-KO) mice were treated with either vehicle (Veh) or a high fat-containing diet (HFD) for 16 weeks. Serum HDL (top) and LDL/VLDL cholesterol (bottom) from 5-7 mice/group was measured using ab65390 following protocol instructions. An asterisk (*) means P<0.05 between WT and FXR-KO vehicle group. A pound (#) indicates P<0.05 between Wt and FXR-KO-HFD group. Image obtained from Li G et al., PLoS One, 2012; 7(4): e35895

  • Functional Studies - HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)
    Functional Studies - HDL and LDL/VLDL Cholesterol Assay Kit (ab65390)
    Measurement of total cholesterol, HDL, LDL/VLDL from serum samples. Total Cholesterol (blue), HDL (green), and LDL/VLDL (cream) cholesterol were measured following the kit protocol.
  • ab65390 HDL and LDL/VLDL Cholesterol Assay Kit
    ab65390 HDL and LDL/VLDL Cholesterol Assay Kit

    Rat serum samples were processed according to the protocol. The quantity of total (dilution range 1:10-1:100), HDL (dilution range 1:1-1:10), LDL/VLDL (dilution range 1:1-1:10) and free cholesterol (neat) was measured colourimetrically, in duplicates, after 40 minutes.

  • ab65390 HDL and LDL/VLDL Cholesterol Assay Kit
    ab65390 HDL and LDL/VLDL Cholesterol Assay Kit

    Signal from standard curve was measured colourimetrically over a period of time. Background signal subtracted and each point on the curve represents duplicate values (+/- SD).

References (219)

Publishing research using ab65390? Please let us know so that we can cite the reference in this datasheet.

ab65390 has been referenced in 219 publications.

  • Chang HC  et al. rAAV-CRISPRa therapy corrects Rai1 haploinsufficiency and rescues selective disease features in Smith-Magenis syndrome mice. J Biol Chem 299:102728 (2023). PubMed: 36410433
  • Lee H  et al. Crosstalk between TM4SF5 and GLUT8 regulates fructose metabolism in hepatic steatosis. Mol Metab 58:101451 (2022). PubMed: 35123128
  • Busatto S  et al. Considerations for extracellular vesicle and lipoprotein interactions in cell culture assays. J Extracell Vesicles 11:e12202 (2022). PubMed: 35362268
  • Abbas AO  et al. Citrullus colocynthis Seed Ameliorates Layer Performance and Immune Response under Acute Oxidative Stress Induced by Paraquat Injection. Animals (Basel) 12:N/A (2022). PubMed: 35454193
  • Abu Helal R  et al. Aortic Fibrosis in Insulin-Sensitive Mice with Endothelial Cell-Specific Deletion of Ceacam1 Gene. Int J Mol Sci 23:N/A (2022). PubMed: 35457157
View all Publications for this product

Customer reviews and Q&As

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1-10 of 63 Abreviews or Q&A

Sensitive and Easy to Use

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
This kit is sensitive for the measurement of cholesterol levels in deer mouse serum. And it is easy to use.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 04 2021

assay on 384-w plate works on mouse CSF

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
The assay can be performed on a 384-well plate, which allows to scale down the volume to 1:5 than stated in the manual. Moreover, the assay allows detection of cholesterol in limited amounts of cerebrospinal fluid from single mice.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Dec 02 2020

Rikard Fristedt - Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)

 Good Average 3/5 (Ease of Use)
Abreviews
I have done with all measurements using those kits and everything worked well. Of course, we need to read and understand the flow and procedure carefully beforehand to avoid confusion and mistake.

Just a little confusion about information of the kit, which made me really thought that we can measure all 3 parameters (HDL, LDL, and total cholesterol) for 100 samples for one kit. Unfortunately, it is not. Since I have 100 samples to measure all those 3 parameters, I then have to order more kits.”
Please make this clear from the beginning!

Overall, I am satisfied with the kits.

Rating 1 to 5 (4/5)
Product ease of use 1 to 5 (3/5)
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

MR. Rikard Fristedt

Verified customer

Submitted Oct 28 2019

Total cholesterol from murine plasma samples

 Average Average 3/5 (Ease of Use)
Abreviews
abreview image
This kit offers the possibility of measuring total cholesterol or HDL and LDL/VLDL fractions separately, which is an advantage over having to purchase two different kits.
I used this kit to measure total cholesterol from murine plasma samples obtained by cardiac puncture and centrifugation (4 C, 20 min, 2000 g) diluted 1:10 with PBS. The kit was easy to use and precise when the results were compared with historical records.
However, when I tried the separation step for HDL and LDL/VLDL fractions, I had the feeling that the design of the kit is not perfect as it is very easy to disturb the pellet and get contamination of your HDL fraction.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Sep 07 2018

Oxidized HDL is a potent inducer of adipogenesis and causes activation of the Ang-II and 20-HETE systems in human obese females.

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
I tried many different product to get HDL and LDL/VLDL better and more clear but failed then I bought Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390)to HDL and LDL/VLDL in obese mice serum to detect the effects of obesity on HDL and LDL/VLDL, as recommended in their datasheets and I never had any problem with it. I think its great product to use. I think this is best product to detect HDL and LDL/VLDL during in vivo studies
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 09 2018

Measuring Cholesterol in Zebrafish Livers

 Excellent Good 4/5 (Ease of Use)
Abreviews
abreview image
I used the HDL and LDL/VLDL Cholesterol Assay Kit (Fluorometric) in order to measure total and free cholesterol in zebrafish liver. Overall the assay was successful. I did have some samples outside of the standard curve range meaning I will need to dilute my samples next time I run the assay. I divided all of my standard and sample fluorescence values by a factor of 10,000 prior to developing my standard curve. Attached is an image of my standard curve. From my standard curve I calculated the concentration of cholesterol that I then applied to the cholesterol concentration equation provided in the protocol. Some example data from my control sample is shown below.


Total Cholesterol: 57.7 ug/uL
Total HDL: 60.9 ug/uL
Total LDL/VLDL: 8.14 ug/uL

Free Cholesterol: 53.7 ug/uL
Free HDL :57.3 ug/uL
Free LDL/VLDL: 6.57 ug/uL

Cholesterol Esters: 3.99 ug/uL
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Dec 13 2017

Comparison of mouse liver extraction methods

 Good Excellent 5/5 (Ease of Use)
Abreviews
abreview image
I tried 2 extraction methods for mouse liver samples (from ApoE KO mice fed a high fat diet) that had been snap frozen and stored in -80 freezer.

I followed the Abcam sample preparation for tissue lysates and I also tried a protocol that had been posted by a previous reviewer of the product. See details below (copy pasted from previous reviewer):

For the separation of HDL and LDL/VLDL
1. Use 0.5% NP-40 in water to homogenize the tissue and then combine with the 2X precipitations buffer.
2. Mix 100 µL of sample with 100 µL of 2X Precipitation Buffer (1:1) in microcentrifuge tubes.
3. Incubate 10 minutes at room temperature.
4. Centrifuge at 2,000 x g (5,000 rpm on a bench-top microcentrifuge) for 10 minutes.
5. Transfer the supernatant into new labeled tubes. This is the HDL fraction.
6. Precipitates contain the LDL/VLDL fraction. To measure the LDL/VLDL fraction, centrifuge the precipitate again at 2,000 x g (5,000 rpm on a bench-top microcentrifuge at RT) for 10 minutes and remove trace amount of HDL supernatant carefully. I tissue homogenate you cannot measure the LDL fraction, only HDL!!!! (In tissue culture it is possibale).
7. Resuspend the precipitate in 200 µL PBS. This is the LDL/VLDL fraction.

If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS and repeat the separation procedure from step 2.

To homogenise the tissue I used a precellys 24 homogeniser.

Overall the protocols were similar but I preferred the Abcam one as all the reagents required are supplied within the kit. The alternative method requires NP-40 which is not supplied which would be expected.

From the image uploaded you can see that the 2 methods (Method 1 - Abcam Method 2 - user) produced very similar absorbance values (colorimetic method was used). The samples were diluted 5x to ensure they were within the standard curve.

Having compared the 2 methods I will proceed with using the Abcam method.

Overall this is a straightforward easy to use kit.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 14 2017

Measure HDL cholesterol in supernatant of Huh-7 cells

 Poor Poor 1/5 (Ease of Use)
Abreviews
The kit is not sensitive enough to detect HDL-cholesterol in Huh-7 culture supernatant neither using colorimetric or fluorometric method
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jul 07 2017

HDL and total cholesterol on liver tissue homogenates

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
TISSUE PREPARATION
For total cholesterol
1. Homogenize 10 mg tissue in 200 μl of chloroform:Isopropanol:NP-40 (7:11:0.1) using a micro-homogenizer.
2. Spin the extract 10 minutes at 15,000 x g at RT in a centrifuge.
3. Transfer all of the liquid (organic phase) avoiding the pellet, to a new tube. Air dry the sup at 50°C to remove chloroform for 7min (in a hood).
4. Dissolve dried sup with Cholesterol Assay Buffer (1:1) by vortexing until homogeneous (it is OK if the solution becomes cloudy).

Note: The extraction procedure can be scaled up if larger amounts of sample are desired.

For the separation of HDL and LDL/VLDL
1. Use 0.5% NP-40 in water to homogenize the tissue and then combine with the 2X precipitations buffer.
2. Mix 100 µL of sample with 100 µL of 2X Precipitation Buffer (1:1) in microcentrifuge tubes.
3. Incubate 10 minutes at room temperature.
4. Centrifuge at 2,000 x g (5,000 rpm on a bench-top microcentrifuge) for 10 minutes.
5. Transfer the supernatant into new labeled tubes. This is the HDL fraction.
6. Precipitates contain the LDL/VLDL fraction. To measure the LDL/VLDL fraction, centrifuge the precipitate again at 2,000 x g (5,000 rpm on a bench-top microcentrifuge at RT) for 10 minutes and remove trace amount of HDL supernatant carefully. I tissue homogenate you cannot measure the LDL fraction, only HDL!!!! (In tissue culture it is possibale).
7. Resuspend the precipitate in 200 µL PBS. This is the LDL/VLDL fraction.

If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS and repeat the separation procedure from step 2.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Mar 29 2016

Question

Is it acceptable to use EDTA-plasma with this assay kit (as compared to plasma produced with heparin)?

Read More
Answer

EDTA is not ideal for measurement of HDL/LDL in samples. EDTA plasma has potential disadvantages that have discouraged its use. Inadequate mixing can result in microclots. Also, EDTA osmotically draws water from red cells, diluting the plasma constituents.

Read More

1-10 of 63 Abreviews or Q&A

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