Overview

  • Product name
    MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric)
    See all MitoBiogenesis kits
  • Detection method
    Colorimetric
  • Sample type
    Adherent cells
  • Assay type
    Cell-based (quantitative)
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    For identifying inhibitors and activators of mitochondrial biogenesis in adherent cultured cells. Each kit contains sufficient reagents to analyze two 96-well plates of fixed human, rat, mouse, or bovine cells. This kit utilizes colorimetric detection for use with standard plate readers. An alternate IR version of this kit is available which utilizes LI-COR® near-infrared IRDyes® for detection - MitoBiogenesis™ In-Cell ELISA Kit (IR) (ab110216/MS642).


     


    In-Cell ELISA Kits use quantitative immunocytochemistry to measure protein levels or post-translational modifications in cultured cells. Cells are fixed in a 96-well plate and targets of interest are detected with highly specific, well-characterized monoclonal antibodies and levels are quantified with enzyme-labeled secondary antibodies.


     


    ab110216 MitoBiogenesis™ In-Cell ELISA Kit (ab110216/MS643) is designed to measure drug-induced effects on mitochondrial biogenesis early in the safety screening process. ab110216 MitoBiogenesis™ In-Cell ELISA Kit is a true duplexing 96/384-well assay that ratios both an mtDNA- and an nDNA-encoded protein in cultured or primary cells, and which requires very little sample prep and few overall steps.


     


    Cells (human, rat or mouse) are seeded in 96- or 384-well microplates, and after exposure to experimental compounds for several cell doublings, the levels of two mitochondrial proteins are measured simultaneously in each well. The two proteins are each subunits of a different oxidative phosphorylation enzyme complex, one protein being subunit I of Complex IV (COX-I), which is mtDNA-encoded, and the other being the 70kDa subunit of Complex II (SDH-A), which is nDNA-encoded. Complex IV includes several proteins which are encoded in the mitochondrion, while the proteins of Complex II are entirely encoded in the nucleus. Optionally, total protein levels can also be measured.


     


    LI-COR®, Odyssey®, Aerius® and IRDye® are registered trademarks of LI-COR Biosciences Inc


     


    Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.

  • Notes

    Related products

    Review the mitochondrial assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Tested applications
    Suitable for: In-Cell ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 2 x 96 tests
    100X Triton X-100 1 x 1.5ml
    10X Blocking Buffer 1 x 15ml
    10X Phosphate Buffered Saline 1 x 100ml
    1X AP Development Solution 1 x 24ml
    1X HRP Development Solution 1 x 24ml
    200X Primary Antibodies 1 x 0.1ml
    2500X AP-labeled Secondary Antibody 1 x 12µl
    2500X HRP-labeled Secondary Antibody 1 x 12µl
    400X Tween-20 1 x 2ml
    AP Development Reagent 1 x 139mg
    1X Janus Green Stain 1 x 17ml
    Plate Seals 2 units
  • Research areas
  • Alternative names
    • MS641
    • MS642
    • MS643
    • MS644

Associated products

Applications

Our Abpromise guarantee covers the use of ab110217 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent dilution.

Images

  • Antibody specificity demonstrated by Western Blot. A Western blot of total cell protein (10 µg) from human or rat cultured cells was probed with the primary and secondary antibodies and scanned with a LI-COR® Odyssey® imager. The two mitochondrial proteins targeted by the two primary mAbs were labeled and visualized specifically despite the presence of thousands of other proteins. Furthermore, reduction of mtDNA levels in human Rho0 (mtDNA-depleted) cells, or inhibition of mitochondrial protein translation by chloramphenicol in rat cells both result in specific reduction of COX-I protein while nuclear DNA-encoded SDH-A is unaffected.
  • Quantitative measurement of the COX-I/SDH-A protein expression ratio. At all cell concentrations, a consistent ratio of mtDNA-encoded protein expression (COX-I) to nuclear DNA-encoded mitochondrial protein expression (SDH-A) is observed in untreated cells. Therefore, normalizing COX-I levels to SDH-A levels simplifies data analysis and eliminates the need to perform all tests at the same cell concentration.
  • Antibody specificity demonstrated by immunocytochemistry. Two-color immunocytochemical labeling of cultured cells with the two MS643 primary monoclonal antibodies specific for COX-I and SDH-A. The two antibodies exhibit striking and specific co-localization in the mitochondria, consistent with the known mitochondrial expression of both proteins.
  • Inhibition of mitochondrial biogenesis by chloramphenicol The IC50 of a drug's effect on mitochondrial protein translation can be determined quickly using the MitoBiogenesis™ In-Cell ELISA Kit. In this example, cells were seeded at 6000 cells/well, allowed to grow for 3 cell doublings in a drug dilution series and then the relative amounts of COX-I, and SDH-A were measured in each well. Chloramphenicol inhibits mtDNA-encoded COX-I protein synthesis relative to nuclear DNA-encoded SDH-A protein synthesis by 50% at 8.1 µM.
  • Cells are grown to ~80% confluency in a 96- or 384-well plate, a drug/other treatment is applied to stimulate a cellular response. The cells are then fixed and permeabilized, effectively "freezing" them. Primary antibodies are then added which bind to their intended targets within the mitochondria or other subcellular compartment. After incubation, the unbound primary antibodies are washed away and secondary antibodies are added. These secondaries are conjugated to either IRDyes® or to an enzyme label (HRP or AP) for the colorimetric versions of the assays. Unbound secondaries are washed away, reaction buffer is added for the colorimetric assays, and the signal is read on a suitable instrument for the kit type.
    » In-cell ELISA diagram in PDF format

Protocols

References

This product has been referenced in:
  • Khdour OM  et al. Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich's ataxia. Bioorg Med Chem 26:3359-3369 (2018). Read more (PubMed: 29773347) »
  • Wang JL  et al. Antifibrotic role of PGC-1a-siRNA against TGF-ß1-induced renal interstitial fibrosis. Exp Cell Res 370:160-167 (2018). Read more (PubMed: 29913155) »
See all 9 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

reliable data

Excellent Excellent 5/5 (Ease of Use)
Abreviews
We use HepG2 and H9C2 cells for assay, and 384-well format, the plate wahser can improve the assay window and performance a lot. Two key factors for assay, cell status and wash step.

Abcam user community

Verified customer

Submitted Mar 27 2017

Dr. Andaleeb Sajid

Excellent Excellent 5/5 (Ease of Use)
Abreviews
I am using this kit for high throughput screening of more than 500 compounds. The kit is highly reproducible and I did not observe any lot to lot variation. I would highly recommend this kit.

Andaleeb Sajid

Verified customer

Submitted Jul 29 2015

Answer


(1) Which human and rat cell lines were used for the testing?
A couple different cell lines were used to generate the data. The cell type used for
Figure 1 were HepG2 cells
Figure 2a. 143B osteosarcoma wild type, 143B Rh0 osteosarcoma (mito depleted), H9C2 Cardiomyocyte Untreated, and H9C2 Cardiomyocyte 10 uM Chloramphenicol
Figure 2b and 3. HeLa cells

(2) How long was the incubation time with chloramphenicol? Was it 7 or 14 days?
This is the protocol we used for culturing the cells for Fig. 1 in 96 well plates.
i. Seed 6,000 HepG2 cells per well (50 uL); in amine coated 96 well plate; avoid outer edge well of plate to reduce any chance of edge effects
ii. Allow cells to attach for minimum of 6 hrs or overnight
iii. Add 50 uL media with 2x Chloramphenical concentration to each well (final 1x concentration @ 100 uL/well); remember to have untreated control
iv. Incubate cells in tissue culture humidified CO2 incubator for 6 days
v. Perform assay according to protocol

(3) Is there a difference seen between RT-PCR and this assay?
To the best of our knowledge we did not compare the ELISA results with RT-PCR results.
However taking into account the mode of action of chloramphenicol it is highly unlikely that mtDNA or mtRNA would have been affected. Chloramphenicol affects translation of mitochondria and not transcription, hence the advantage of using antibodies rather than the PCR method. The PCR method will miss any OXPHOS effects due to translational toxicity.

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Answer

Thank you for your phone call today. Please find the reply from the lab below, and let me know if you have any further questions.

In our experience it is necessary to treat cells for a few passages to observed the effect on mitochondrial protein translation. If the customer is concern about having viability changes that can confound the results, they could titrate chloramphenicol so they find the minimal amount of compound that can affect their cell system for at least 3 passages.

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Question


I have been using the IR kit (ab110216) and using Chloramphenicol and ddC in HepG2 cells as my control for mitochondrial tox. I have also attempted this with the Colormetric kit (ab110217).

The HepG2 cells were obtained from ATCC and grown in EMEM(ATCC) +10% FBS + 1%P/S

The Chloramphenicol was obtained from Selleck Chem (http://www.selleckchem.com/products/Chloramphenicol(Chloromycetin).html) 100mg diluted in DMSO to 100mM Stock.

ddC was obtained in house at a stock concentration of 100mM DMSO.

Assay Conditions: Complete growth media in a final conc of 0.50%DMSO, 3-fold, 8pt serial dilution with top concentration of 100uM, all compounds tested in triplicate with Janus Green Stain performed after readout and another duplicate plate in parallel readout with 50% Cell Titer Glo.

Seeding Density: Tried 3000c/w and 8000c/w for 7days post drug addition.

Equipment: I use the LICOR Oddysey for IR readout, and Omega



So I have a few questions:

1. On the scientific support website, HepG2 cells were recommended to be seeded at 1500-3000c/w. In pg 13 of the IR kit protocol (attached), cells were seeded at 3000c/w (Su confirmed they were HepG2) for 3 cell doublings (which I assume to be about 5-6 days) and Chloramphenicol was seen to reduce COX-1 relative to SDH-A with a CC50˜3.5uM. On pg 14-15 of the Fluorescent kit protocol (attached), cells were seeded at 6000c/w for 6 days and looks to have about the same CC50 with Chloramphenicol of about 3uM. On pg 17 of the colormetric handbook, cells were seeded at 6000c/w for 3 cell doublings and Chloramphenicol had a CC50 of 8.1uM. My question is what seeding density should I be using for HepG2 if I need to incubate with serially diluted compound for 7 days (one time only, no re-dosing)? The seeding should not vary by kit I presume?

2. Also, I have not seen the same effects on the HepG2 cells with Chloramphenicol treatment after 7 days, when seeding at 3000c/w or 8000c/w (data shown below). What is the source of your drug, final % DMSO used, what was your compound dissolved in and what was your highest treatment?

3. On pg 2 of the biogenesis playbook (attached), the chart shows that human HepG2 cells were treated for 5 population doublings with antibiotics and 40uM Chloramphenicol produced about 70% inhibition of mitobiogenesis at the endpoint. What was the seeding density and endpoint in hours (for 5 population doublings), and what was the source of the Chloramphenicol?

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Answer



Looking at your data the results do look pretty good and the kit is performing as expected. Looking over the your questions I would like to emphasis that these kits are developed at different times so there are slight variations in experimental procedure and conditions aren’t specific to each kit. As for the direct questions asked in their query, see below:



My question is what seeding density should I be using for HepG2 if I need to incubate with serially diluted compound for 7 days (one time only, no re-dosing)? The seeding should not vary by kit I presume?Using HepG2 cells for a 7 day treatment we initially seed cells at 3000 cells per well.
Kits have been developed independently and at different times, therefore conditions aren’t necessarily the same between kits.



What is the source of your drug, final % DMSO used, what was your compound dissolved in and what was your highest treatment?



We used chloramphenicol from Sigma
Final Concentration of DMSO was 0.2%
Chloramphenicol stock used was 10 mM prepared in DMSO
Treatment ranged using a 2-fold serial dilution curve highest concentration was 64 micromolar down to 0.25 micromolar, and an untreated group





What was the seeding density and endpoint in hours (for 5 population doublings), and what was the source of the Chloramphenicol?In our hands we determined population doubling to about 30-33 hours for hepg2 cells.
Chloramphenicol was sourced from sigma

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Answer

we suggest to perform a dilute acid wash step: after fixation and before permeabilization, incubate cells for 5 min in 0.5% acetic acid in 1X PBS, followed by a PBS wash. Then proceed to permeabilization and the rest of the protocol. The acetic acid should inhibit the endogenous AP activity without affecting antibody binding. This step has been added to an updated protocol booklet that can be downloaded through the abcam webpage, previous versions of the protocol booklet lack this step which can greatly improve the signal to noise ratio.
performing the Janus green staining to determine relative cell density per well:
If you decided to do this staining it is important to make clear that Janus green is very robust and in our experience requires thorough washing to remove excess dye from the wells. Rather than rely on a pipetting of ultrapure water to wash the plate, you can completely submerge the plate in a large container filled with clean ultrapure water for 1 min, remove plate and empty excess water by inversion and gently shaking into a waste container. Repeat this washing 5 times with clean ultrapure water each time, then blot dry and proceed with HCl treatment.

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Question
Answer

Malheureusement, ils utilisent principalement ce kit pour étudier l'inhibition de la réplication ADN mitochondrial. Cependant, ce kit pourrait en théorie être utilisé pour mesurer l'augmentation de l’ADN mitochondrial. Il existe de nombreuses études qui ce concentre sur des molécules qui peuvent activer la voie PGC1a. PGC1a est une protéine qui active la biogénèse de la mitochondrie. Il faudrait regarder dans la littérature pour voir quelles molécules ont déjà été utilisées pour activer cette voie.

Sur la fiche technique d’ab110217 nous avons une image de l'immunocytochimie (ICC). Les anticorps utilisés sont :

Anti-MTCO1 antibody [1D6E1A8] - Mitochondrial Marker, ab14705 (qui dans ce kit est détecté avec HRP) et Anti-SDHA antibody [2E3GC12FB2AE2] - Mitochondrial Marker, ab14715 (qui dans ce kit est détecté avec AP).

Vous pouvez achetez ces anticorps séparément, ce qui est recommandé pour obtenir les meilleures résultats en immunofluorescence, sinon vous pouvez utiliser le kit ab110217 et utiliser les anticorps primaires 200X. Notez que l’image sur la fiche technique n’utilise pas ce kit mais les anticorps individuels. Ce kit n’est donc pas garantis de fonctionner en immunofluorescence.

Nous avons d’autres kit au catalogue qui test le métabolisme de la mitochondrie qui pourrait être intéressant pour vous :

Luminescent ATP Detection Assay Kit (ab113849)
TMRE—Mitochondrial Membrane Potential Assay Kit (ab113852)
JC1 - Mitochondrial Membrane Potential Assay Kit (ab113850)
DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit (ab113851)
Extracellular O2 probe (ab140097)
Extracellular O2 probe (with mineral oil) (ab140098)
Extracellular O2 probe (with high-sensitivity oil) (ab140099)
Glucose Uptake Assay Kit (Colorimetric) (ab136955)
Glucose Assay Kit (ab65333)

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Answer



"The pH should be 9.8. We can send QC data to the customer if we know the exact lot number (letter followed by 4 digits) written on the label of the tube. We have checked all the buffers made in the last year and without exception they all have been made to pH 9.8. It is very puzzling the lack of signal on both the AP and HRP channels (as described below). Typically, in the absence of a quenching solution there should be at the very least some background signal on both channels. Furthermore, the HRP readout in control samples is extremely robust, so the lack of signal in this channel even in the control sample makes me believe that there may not be enough cells at the bottom of the wells. I asked before whether the customer could send the data, including the settings used to read the assay (end point – time of incubation or kinetic).
We have performed this assay many times on HepG2 cells, which are the cells the customer has in hand. However, for treatments I know by experience it is not possible to grow the cells in a 96 well plate for more than 1 week. The customer is doing experiments for 2 weeks. It is therefore essential to get the Janus green data to compare this with our records.
Until we receive the lot numbers, my suggestion to the customer for now is to ensure the HepG2 cells are seeded at no less than 4000 cells per well on growth media in a 96 well plate. They should be allowed to adhere overnight and treatments should be initiated the following day. Media (containing compounds or diluent) should be changed every other day and assay should be performed no later than 7 days after initiation of treatment. For longer treatments, my suggestion is to treat the cells in bulk on a 100 or 150 mm dish to allow more passages for the cells. At the end of the treatment, cells should be trypsonized, seeded on a 96 well plate at a single density (about 25k – 50k/well), allowed to adhere for a few hours and fixed."

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Answer



Looking over the data set it appears that the HRP signal has a good signal:background ratio. With regards to the AP signal, it does appear to have high background in control wells. We suggest performing a dilute acid wash step: after fixation and before permeabilization, incubate cells for 5 min in 0.5% acetic acid in 1X PBS, followed by a PBS wash. Then proceed to permeabilization and the rest of the protocol. The acetic acid should inhibit the endogenous AP activity without affecting antibody binding.

The Janus green stain is very robust and in our experience requires thorough washing to remove excess dye from the wells. Rather than rely on pipetting of ultrapure water to wash the plate, you can completely submerge the plate in a large container filled with clean ultrapure water for 1 min, remove plate and empty excess water by inversion and gently shaking into a waste container. Repeat this washing 5 times with clean ultrapure water each time, then blot dry and proceed with HCl treatment.

Read More

1-10 of 18 Abreviews or Q&A

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