i can't get well mitochondria staining pattern when staining my cell line with Mitotracker Red CMRos from Invitrogen. Does anyone have any solutions?
The picture you have made open seems to be diffusely stained. In my experience, Mitotracker probe is easy to stain mitochondria in live cells in 10-15 minutes in the 37-degree incubator. There are possible two points in which you may revise.
1) To observe and take a picture after washing cells with PBS twice or three times.
You should not observe the cell in the colored medium with serum, otherwise the staining would be diffuse in the microscope.
2) To filtrate the liquid containing Mitotracker probe. There are several Mitotracker probes, some of which tend to crystalized.
I disagree that mitotracker probes, especially CMXRos, need to be washed or filtered. The probe should be dissolved in DMSO at a stock concentration of 1 mM, in that condition it should not crystallize. Also, the accumulation of the probe in the mitochondria so be so strong that it should drown out any background fluorescence, negating the need for washing.
A few questions to help troubleshoot:
- What cell type are you using?
- What concentration are you using?
- What is your protocol for staining? (times, reagent addition order, etc)
- Are the cells alive when you add the stain? When you image?
- What are your filter sets for imaging? Or, if laser-based, what is your excitation line?
Thanks, Jordan. the cell i stained is a kind of neuroblastoma cell lines. what's more, the concentration i am using is 100nM. Do you think it is too low? At this concentration, i stained the alive cells for 10-50 min (i have tried various staining time in this range).Then i washed the cells for 3 times with PBS. Following is the fixation with 4%PFA for 15 minutes. after that, i washed the cells for 3 times with PBS. subsequently, DAPI was used to stain the nuclei. Do you think the washing after mitochondria staining will lead to the leakage of the dye?
Why not try TMRM? I use this routinely and it has never failed. Otherwise do a dose/stain trial. Start with 100nM and go up in serial dilutions, use a 96 well plate or something small so you are not using too much dye. Also it could be your fixation. Try imaging the stained cells live and then fix. image them again once fix. You should be able to find the problem. Confluency of cells should not affect the stainning.
I have found that when I fix cells I need to add about 20-50% more MitoTracker CMXRos for the staining the be maintained well through subsequent washing steps, but this may be due to my particular cell type (macrophages). For live imaging I usually use 100 nM, for fixation I usually use 150 (and rarely but occasionally 200 nM). 30-45 minutes incubation with the stain should be sufficient.
I would recommend that you at least test the cells by imaging them while they are alive and viable to see that the stain is being accumulated in the mitochondria at all prior to fixation. If you do not have mitochondrial staining in live cells, you obviously won't have it when they are fixed.
Do not use PFA. Try formalin. Stain the cells by mitotracker for 15-30min. Dilute 37% formalin to 3.7% by medium and warm to 37oc. Fix for 5-15min at 37oc. Wash and stain by DAPI.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology