MitoBiogenesis™ In-Cell ELISA试剂盒(Colorimetric) (ab110217)

(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.

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.

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

Some cell lines may have low level of SDHA – possibly longer incubations may help.


Though not ideal – the levels of COXI could be normalized to the included cell stain (Janus green).

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.

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)

"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."

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.