Mitochondria Isolation Kit for Cultured Cells (ab110170)

The level of contamination depends on the quality of the sample. To minimize contamination, I suggest to perform the isolation of freshly isolated/harvested cells. Any freeze/thaw step of the cell pellet will not yield a good preparation. However, there will always be a small level of contamination from other membrane organelles.

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.

You can use the leftover reagents of kit ab110170 with tissues. However, you will need to follow the tissue protocol to get the best outcome.

Note, we have a specific Mitochondrial isolation kit for tissue: ab110168.

Large clearance pestle (A)[clearance 0.0030-0.0050 in] is used for the initial sample reduction, such as tissue prep;

Small clearance pestle (B) [0.0005-0.0025 in] is used to form the final homogenate.

When prepare cell homogenate, we recommend to use pestle B directly to gain a fine final prep.

The procedure can accomodate approximately 20 benchtop mitochondria isolations, assuming about 4x10^7 cells per isolation are used. These amounts can be scaled up or down accordingly, as long as the cells are resuspended to 5 mg/ml in Reagent A, and the same volume of Reagent B is also used as indicated in the protocol. Then, you can adjust the amount of reagent C you resuspend the mitochondria in based on your desired end concentration of mitochondria.

ab109719 is not suitable for this purpose, this kit is based on detergent fractionation of cellular proteins into three fractions. RNA distribution was not tested with this kit.



Mitochondrial isolation kits (ab110168 – ab110171) are designed to isolate whole mitochondrial organelle, as such these kits are suitable to isolate mRNA that is attached to/ within this compartment. I would recommend to supplement all the buffers of the Mitochondrial Isolation Kit with RNAse inhibitors. We do not have any data regarding the yield of mRNA. The reference below maybe useful reading:



http://www.ncbi.nlm.nih.gov/pubmed/6197615

Attardi G, Montoya J.

Methods Enzymol. 1983;97:435-69. No abstract available.

PMID:

6197615



Please let me know if you have any further questions!

I can confirm that the mitochondria isolated using this kit can be used in other biological assays.
Please check protocol booklet page number 2 for expected yield based on cell line:
Sample / Starting Material / Expected Yield
MRC-5 / 4 – 150 mm plates / 0.5 mg*
HepG2 / 4 – 150 mm plates / 1.0 mg*
HDFN / 2 – 150 mm plates / 0.5 mg*

You will want to use either ab110170 or ab110171 (which includes the homogenizer), as these are optimized for isolating fractions from cultured cells versus tissue samples. The supernatant from SPIN2 is the cytosolic fraction, though it might be contaminated with nuclear protein depending on what percentage of nuclei were lysed during the homogenization. The aim is to lyse the plasma membrane by weakening them with the freeze/thaw treatment followed by homogenization, but without damaging the nuclear membrane. The pellet after SPIN1 is ideally intact nuclei.

For the freeze/thaw, we recommend pelleting the cells, aspirating most of the medium, then dipping the vial into liquid nitrogen for one minute and then 37C for one minute or until thawed. The pellet can be stored at this point at -20C or -80C, whichever is more convenient. When you are ready to continue the isolation protocol, add reagent A as instructed in step 3 of the protocol (ab110170). If you do not have liquid nitrogen, store the pellet at -20C or preferable at -80C. When you are ready to isolate mitochondria from all of your samples, thaw the pellets at 37C for one minute to thaw and continue by adding reagent A, step 3.

We do not have data for stability of samples stored at -20 or -80C. Stability of proteins or other biomolecules will depend on what you are investigating, but for the purposes of isolating fractions, we expect this kit should effectively isolate mitochondria from samples stored for several months, if not longer.