I have been attempting to stain LN229 glioblastoma and MSU1.1 human fibroblast cells with the mitochondrial membrane potential dye (JC-1). The dye localizes to mitochondria using the potential gradient and depending on JC-1 localized concentration, will emit red (large membrane potential) to green (low potential) light.The dye is considered a "living" dye and requires the cell to be alive to properly work and also prevents fixing with formaldehyde.
So far I have had cytotoxicity problems, my guess to high a concentration of DMSO; it is now at recommended .1% DMSO concentration, as well as keeping the cells on the coverslip and mounted onto a microscope slide. Any tips, tricks, or protocols would greatly help.
Protocol:
0.5 ml of diluted cells in media is added to a cell plate well containing a Poly-L-lysine coated coverslip and is incubated for 3 days at 37C and 5% CO2. Following the 3 day period, the media is removed and 200uL of JC-1 dye diluted in culture media is added:
Different used dye-media solutions:
(10ug JC-1, 5% DMSO) in culture media
(10ug JC-1, 1% DMSO) in culture media
(5ug JC-1, .1% DMSO) in culture media = current
Incubated for 15 minutes at 37C and 5% CO2
Dye media is removed and 0.5 ml of 1X PBS is added for 1 min and removed.
The coverslips are then removed and placed on a drop of PBS on top of a glass slide and mounted with nail pollish. Ever step involved the JC-1 is performed at minimal light (lights turned off) or in a dark room.
When looking at the slides, it seems that many of the previously adhered cells have "washed off" or have lysed do to cytotoxicity, which with the current DMSO concentration should not happen.
Would DMSO or PBS cause the cells to detach from the coverslip?
Do adhered cells fall of easily?
Hi Walter Valdez,
we use JC1 for FACS analysis of mitochondria. However, I recognize several issues:
ad1.) Cells or Mitochondria need to be energized to develop J-Aggregates. Airtight mounting results in hypoxia and collapse of ΔΨm. JC1 does (only) stain living cells/mitochondria.
ad2.) If your PBS is Ca2+-free, Cells will not adhere
ad3.) PBS is a poor medium for this analysis, because of stressing your cells
ad4.) We dissolve JC1 in DMSO, too. Because of its high reducing capacity, we perform further dilution with PBS to achieve a reduced DMSO concentration
ad5.) Since JC1 accumulates in living cells, I recommend to omit your final washing step
With best regards
SPS
Hi Walter Valdez,
we use JC1 for FACS analysis of mitochondria. However, I recognize several issues:
ad1.) Cells or Mitochondria need to be energized to develop J-Aggregates. Airtight mounting results in hypoxia and collapse of ΔΨm. JC1 does (only) stain living cells/mitochondria.
ad2.) If your PBS is Ca2+-free, Cells will not adhere
ad3.) PBS is a poor medium for this analysis, because of stressing your cells
ad4.) We dissolve JC1 in DMSO, too. Because of its high reducing capacity, we perform further dilution with PBS to achieve a reduced DMSO concentration
ad5.) Since JC1 accumulates in living cells, I recommend to omit your final washing step
With best regards
SPS
Hi Reem A.m
attached you´ll find our protocol as described earlier:
"Flow cytometric analysis of mitochondrial membrane potential (ΔΨm).
In presence of rotenone and succinate 100 µg of mitochondria were suspended in 100 µl Buffer 5 and energized by 10 mM succinate after inhibition of complex I by 2 µM rotenone. Mitochondria were stained by 2 µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC1, Enzo Life Sciences GmbH Lörrach, Germany). ΔΨm-stability was assessed after CCCP-induced (0.5 µM) mitochondrial uncoupling. Fluorescence-activated cell sorting (FACS) was performed using a BD FACScan Flow-Cytometer (Becton Dickenson Heidelberg, Germany) as described by Lecoeur (7). Briefly, settings of FACScan were applied as followed: Mitochondrial size was measured by the forward scatter (FWS) adjusted to an E-00 setting with logarithmic amplification of 5.41. Mitochondrial granulation was detected by the sideward scatter (SSC) adjusted to linear amplification at a voltage of 581 and a gain of 4.27. JC-1 green fluorescence reflects JC-1 monomers and was detected using the FL-1 channel. Formation of J-aggregates led to orange fluorescence measured in the FL-2 channel. Fluorescence was recorded using the logarithmic amplifier mode. FL-1 was adjusted to a voltage of 907 mV and 622 mV for FL-2, respectively. Spectral compensation was performed as follows: FL1 – 18.5% * FL2 and FL2 – 25.4% * FL1. 25000 counts of the main mitochondrial region gated on FSW / SSC parameters were recorded. Data were analyzed using the WinMDI software (http://facs.scripps.edu/software.html). The ratio of J-aggregate+-mitochondria and JC1+-mitochondria was used to assess ΔΨm. Measurements were performed in native mitochondria, immediately after JC 1 staining, 10 min after JC 1, immediately after uncoupling with CCCP and 1,2 and 3 min after CCCP.
7. Lecoeur H, Langonne A, Baux L, et al.: Real-time flow cytometry analysis of permeability transition in isolated mitochondria. Exp Cell Res 2004;294:106-17."
I hope you will succeed with your analysis.
With best regards
Sebastian Sommer
Consider staining with the Rhodamin 123 dye. It has several advantages when compared to JC1. ΔΨm is represented by only one wavelength and it is more specific than JC1.
Rhodamine 6G is another dye you could try, but a regular TRITC filter set will not see it properly so you need to be careful. This is true of all these dyes - if you don't have the proper filters, you won't see anything, or you'll see the wrong thing. MitoTrackers (Life Technologies, I think) come in different colors so you can pick one to match the filters you have.
Hellow,
How long can I store the stock solution of JC-1 in -20oC?
Notes
- A 6-, 12-, 24-, or 96-well culture plate can be used for this method. It is recommended that the cell be no more than 80% confluent by the time of visualization.
- JC-1 staining solution is difficult to prepare in aqueous medium due to its low solubility and tendency to form particulates that are difficult to eliminate, so make sure JC-1 reagent is completely thawed before diluting it into culture medium.
- All staining procedures must be performed without direct exposure to intense light, as JC-1 is light sensitive.
- If staining is too intense, bring down the final JC-1 concentration may be required.
- It is imperative that samples be analyzed immediately following completion of the staining.
- Buffer or media must be present in the wells during reading of the signal. Don’t allow wells to dry out.
More details：
https://www.creative-bioarray.com/support/jc-1-mitochondrial-membrane-potential-assay.htm
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