MitoPT™-TMRM
Assess mitochondrial permeability and depolarization events
ImmunoChemistry Technologies' MitoPT™-TMRM assay kit offers a straightforward method of assessing mitochondrial depolarization via flow cytometry, fluorescence microscopy, and fluorescence plate reader assay formats.
MitoPT - TMRM (100 Tests)
MitoPT - TMRM (500 Tests)
The MitoPT™ potentiometric dyes exhibit very low toxicity and display rapid and reversible membrane equilibration properties. Inside a healthy, non-apoptotic cell, the lipophilic TMRM dye, bearing a delocalized positive charge, enters the negatively charged mitochondrion where it accumulates in an inner-membrane potential-dependent manner. When the mitochondrial collapses in apoptotic or otherwise depolarized cells, the MitoPT™ TMRM potentiometric dye no longer accumulates inside the mitochondria and becomes evenly distributed throughout the cytosol. When dispersed in this manner, overall cellular fluorescence levels drop dramatically and this event can easily be visualized by fluorescence microscopy or quantitated by flow cytometry or fluorescence plate reader analysis techniques. The MitoPT™-TMRM assay easily distinguishes between non-apoptotic cell populations and those cell populations that are transitioning into an apoptotic state.
The MitoPT™-TMRM kit can be used in conjunction with your existing research protocols. Grow your cells using your usual cell cultivation protocol. If using an apoptosis or oxidative stress induction model system, simply induce apoptosis according to your existing procedure. Once you have induced apoptosis in your cells, spike enough MitoPT™ dye solution into each test and control sample to give a 20-200nM dye concentration. Incubate the cells for 20 minutes at 37C to allow the MitoPT™ potentiometric dye to equilibrate within the polarized mitochondria. If the cells are not undergoing some form of metabolic or apoptotic stress, the mitochondrial membrane will remain intact, resulting in the concentration of the MitoPT™ dye within the negative/alkaline environment of the mitochondrion. If the cells are apoptotic, the mitochondrial membrane will depolarize, causing the MitoPT™ dye to be dispersed throughout the cytosol. This MitoPT™ dye dispersion results in a dramatic reduction in the overall fluorescence of the affected mitochondria in particular, and the entire cell in general. MitoPT™-TMRM excites optimally at 548 nm, but also yields excellent results using the common Argon blue line (488nm) laser of most flow cytometers. Optimal emission from the MitoPT™-TMRM reagent lies in the FL2 emissions region (573 nm). Orange emission filters (570nm +/- 10 nm) can be utilized to detect the presence of these potentiometric dyes using flow cytometry-based protocols.
MitoPT™-TMRM Depolarization Detection Kits can evaluate apoptosis using flow cytometry, fluorescence microscopy, or a fluorometric plate reader. When cells stained with MitoPT™-TMRM are run through a flow cytometer, the instrument will measure apoptosis by monitoring the loss of orange (573 nm) fluorescence intensity (FL2 emission spectrum) relative to the normal (negative) control population. Apoptotic cell mitochondria have a reduced charge that results in lower levels of the potentiometric dye within these organelles. TMRM has been used concurrently with other fluorophores in multi-parametric, flow cytometry based analyses measuring mitochondrial membrane depolarization, caspase activation, phosphatidyl-serine exposure, and/or cell viability within a single cell population. When cells stained with MitoPT™-TMRM are analyzed with a fluorescence plate reader, the instrument will measure apoptosis by monitoring the amount of emitted orange fluorescence. Healthy control cells bearing mitochondria with normal electrochemical gradients will concentrate the potentiometric dye to a greater extent than will apoptotic cell populations. The difference in fluorescence output of these two populations can be easily distinguished on a fluorescence plate reader using 540 +/- 10 nm excitation / 570 nm +/- 10 nm emission filter tandems in black 96-well plates. Analysis of MitoPT™ TMRM stained cells using fluorescence microscopy will reveal non-apoptotic cells containing bright orange fluorescent mitochondrial bodies (TMRM dye accumulates within healthy mitochondria). In contrast, apoptotic and metabolically stressed cells will have noticeably fewer bright fluorescent mitochondria and more dim or completely non-fluorescent mitochondria. The overall brightness of these cells will also be visibly reduced as a result of the mitochondrial depolarization event.
Following the flow cytometer and fluorescence microscope protocols, each sample can be stained in a 0.5-1.0 mL cell culture volume containing from 20-200nM MitoPT™ dye concentration, depending upon user requirements for cell brightness, and if a wash step is aceptible. Protocols using MitoPT™ TMRM at concentrations > 50 nM should generally include a single wash step to minimize background fluorescence issues. The MitoPT™ 500 Test Kit contains enough TMRM reagent to evaluate 500 flow cytometry or fluorescence microscopy cell samples at a 200 nM dye concentration. If using a fluorescence plate reader analysis protocol, each 1 mL cell culture sample requires a concentration of 100-200 nM MitoPT dye. Accordingly, the MitoPT™ 500 Test Kit will process 500 samples spiked at a 200nM dye concentration, or 1000 samples spiked at 100 nM dye concentration.
Fluorescence microscopy analysis of mitochondrial membrane depolarization in Jurkat cells. Cells were treated with or without 1 staurosporine for 2 hours at 37C and then labeled with MitoPT™-TMRM for 20 minutes at 37C. Collapse of the mitochondrial membrane potential is indicated by a decrease in the mitochondrial fluorescence output, associated with the onset of apoptosis or other depolarizing event.
Kit contains:
- MitoPT-TMRM reagent
- CCCP
- Assay buffer
Sample staining protocol for suspension cells:
- Culture cells up to 1 x 10^6 cells/mL.
- Induce apoptosis or mitochondrial depolarization following your experimental protocol.
- Reconstitute the reagent with DMSO to form the stock concentrate (can be frozen for future use).
- Dilute stock concentrate with 1X Assay Buffer to form the working solution.
- Add 10mcL of the working solution directly to a 1mL aliquot of your cell culture for labeling.
- Incubate 10-15 minutes.
- Wash cells twice.
- Analyze data using a fluorescence microscope, plate reader, or flow cytometer.