MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

On our datasheet we state that this kit can be used with human and cow.

Therefore yes I can confirm that you can use it on your human samples. What I think may be confusing is that we supply bovine heart mitochondria rather than human.

To isolate mitochondria from human PBMCs I suggest you use the Mitochondria Isolation Kit for Cultured Cells (ab110170) (https://www.abcam.com/ab110170) and start with a minimum of 8x10^7 PBMC cells for a yield of 1mg. The mitochondria from PBMCs may be frozen prior to detergent extraction. The higher the mitochondrial preparation protein concentration, the longer the stability at -80C storage. The standard mitotox kits are shipped with frozen whole bovine heart mitochondria at 5.5mg/mL.

I suggest you do side by side detergent extraction of the bovine heart mitochondria with the PMBC mitochondria and use the BHM mitos in at least 2 or 3 wells of the plate to ensure that the assay is working.

I do not recommend using this kit with whole blood.

It is crucial that this type of data is analyzed based on the actual kinetic trace (via softmax software). When a reduction trace is superimposed you will notice that the kinetic change in signal is not constant for the entire assay. The speed of change will vary over the course of the read. Activity assays should be measured in the region where the maximum drop occurs. Most software is capable of adding a lag and an end phase to the analysis. When this is applied, maximum mOD/min is calculated by the software. This allows to have greater reproducibility.

The mitotox kits will work with mitochondria from any human tissue. Therefore it will work with mitochondria from human PBMCs.


To isolate mitochondria from human PBMCs I suggest the customer to use ab110170 (Mitochondria Isolation Kit for Cultured Cells) and start with a minimum of 8x10^7 PBMC cells for a yield of 1mg. The mitochondria from PBMCs may be frozen prior to detergent extraction. The higher the mitochondrial preparation protein concentration, the longer the stability at -80ºC storage. The standard mitotox kits are shipped with frozen whole bovine heart mitochondria at 5.5mg/mL. I suggest the customer to do side by side detergent extraction of the bovine heart mitochondria with the PMBC mitochondria and used the BHM mitos in at least 2 or 3 wells of the plate to ensure that the assay is working.


I do not recommend using the MitoTox kits with whole blood.

The kits that are the best suited for your experiment are listed below. I am having our datasheets team update the species reactivity.
ab109903, Complex I; reactivity = Human, mouse, rat, bovine
https://www.abcam.com/mitotox%E2%84%A2-complex-i-oxphos-activity-microplate-assay-ab109903.html
ab109904, Complex II, reactivity = Human, mouse and bovine
https://www.abcam.com/mitotox%E2%84%A2-complex-ii-oxphos-activity-microplate-assay-ab109904.html
ab109905, Complex II + III, reactivity = This is an in solution assay so it will work with mitochondria from any species, provided that the mitochondria is of good quality. It will not work with homogenate – it requires purified mitochondria
https://www.abcam.com/mitotoxtrade-complex-ii-nbsp-iii-oxphos-activity-microplate-assay-ab109905.html
ab109906, Complex IV, reactivity = Human and bovine
https://www.abcam.com/mitotoxtrade-complex-iv-oxphos-activity-microplate-assay-ab109906.html
ab109907, Complex V, reactivity = Human, mouse, rat and bovine.
https://www.abcam.com/mitotoxtrade-complex-v-oxphos-activity-microplate-assay-ab109907.html

ab110419, Complexes I - V (contains the five kits above at a discounted price)
https://www.abcam.com/mitotox%E2%84%A2-complete-oxphos-activity-assay-kit-5-assays-ab110419.html
In regards to inhibitor treatment, the laboratory agrees that the effects would be reversible. See their response and recommendations below:

If you were to treat the cells with for example rotenone, then isolate the mitochondria from rotenone treated cells, this will wash off all the rotenone from the sample during the sample prep and would simply find no inhibition detected by the assay.

What we would recommend is to test normal mitochondria in-vitro with the inhibitor compound as it is suggested in the kit protocols above.
The kits have a bovine heart mitochondria control, which you can use to see how the assay works (creating a positive and negative control with BHM plus or minus inhibitors).

Then you can compare the BHM results with the results obtained with the mitochondria prepared from your cell system.
The compound inhibitor must be present during the activity assay, otherwise the data will not reflect the immediate in vitro inhibitory effect of the compound, but more likely the downstream effect of that compound (i.e. If the compound generates as a downstream effect oxidative stress and this in turn affects the activity of one of the complexes of the electron transport chain).

Thank you for contacting Abcam.

MitoSciences is part of the Abcam family now, and I have directly contacted them regarding your inquiry. They responded that the 384 well plate format has not been validated or produced. Since Abcam and MitoSciences are one in the same, they will let me know if anything changes and they are able to produce ab110419 in 384-well plate format. While this is not currently being explored I will inform you if anything changes.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for contacting Abcam.

I contacted the supplier, and they responded that ab110419 has not been validated in 384 well plate format.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for contacting Abcam.

We only make custom products for bulk orders of 10 or more products. If you would like to inquire about placing a bulk order for ab110419 in 384-well format please reply to this email, and we will see if the supplier can produce the kit in the format you desire. If the supplier will produce ab110419 in 384-well format, we will send you back an invoice specific for your custom bulk order.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for contacting us.
The assays measure the direct effects of drugs on the 5 OXPHOS complexes and can be used on human samples.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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I now have further information to share with you.

The antibodies used in the MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays), ab110419,to coat the plates are cross-reactive againstthe human proteins. The kit can therefore be used with Human mitochondria, however the signal will be better with bovine heart mitochondria.

My colleague at Mistosciences has suggested that you could test the compounds in vitro with the bovine mitochondria provided as well as with mitochondria from normal human brain and mitochondria from tumor brain.The bovine samples will allowyou to ensure that the kit works as expected.It will also giveyou a reference point in regards to whether the compound inhibit the enzyme or not.
The Mitochondria may be prepared with kit https://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.html. For brain samples, the protocol calls for 0.3 – 0.4g of tissue to expect a yield of 4 – 5mg. Following the protocol, the mitochondria may be frozen at -80°C as whole mitochondria and can be solubilized on the day of the assay side by side with the bovine heart mitochondria provided.
Another option is to use the regular activity kits, however those kits are not optimized to be used with compounds as they do not provide null wells. These null wells are important as some compounds can react with the components of the activity buffer in the absence of enzyme.
https://www.abcam.com/Complex-I-Enzyme-Activity-Microplate-Assay-Kit-ab109721.html
https://www.abcam.com/Complex-II-Enzyme-Activity-Microplate-Assay-Kit-ab109908.html
https://www.abcam.com/ATP-synthase-Enzyme-Activity-Microplate-Assay-Kit-ab109714.html
https://www.abcam.com/Complex-IV-Human-Enzyme-Activity-Microplate-Assay-Kit-ab109909.html



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