We are staining our endothelial cells with the live cell staining calcein. Due to a time-consuming experimental set-up, it would be very helpful if the cells could be fixated after calcein-staining. I haven't found a protocol yet - maybe because this isn't possible at all? Or does somebody else have a protocol for that issue?
Thanks in advance
Works fine, not too much background even in re-seeded tissue.
Thanks!
Hi Mareike and Thomas, I am looking for a similar protocol. I would like to label live cells with a fluorescent dye and I would need the dye to be retained after fixation. Mainly because imaging my plates can take up to 12 hours.
I always thought the Calcein dyes could simply not be fixed and they would leak out, but it seems you had some success. Could you let me know exactly which dye you used at what concentration and how you fixed your cells?
Thank you very much... mario
Hi Mareike and Thomas, I tried fixing cells with 2% PFA, 15' RT and when performing flow cytometry, I see a new peak one log dimmer than the unfixed (see attached pictures, the two samples are identical in terms of staining). Any thoughts?
Any help is appreciated.
Best,
IK
I want to do that as well. I want to stain the dissociated cells with DRAQ5 and Calcein AM and then I want to fix the cells using 1% or 2.5% Formaldehyde and then stop the fixation after 10-15 mins. After that I would like to check and sort the cells using FACS. Do you think if it's possible?
Aparently this will not work according to Fisher " Calcein dyes diffuse into cells, the 'AM' moiety is cleaved by cellular esterases and then are observed in the cytoplasm without binding to anything. This provides a 'whole cell' label. Calcein dyes may be pumped out by normal cellular efflux mechanisms, sometimes within a very short time, especially for cell types that may exhibit drug resistance, unless the efflux is inhibited (such as with probenecid). The dyes are not crosslinked with aldehyde-based fixation, unlike protein-binding CellTracker dyes, and thus will be lost upon fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dyes from the cell."
Can anyone recommend a way to label the whole cell that is retained after fixation? I am trying to threshold to cell area. thanks
Fluospheres (thermoFisher, 0.04um, different colours) are the best: incubate 30min in suspension. cells retain the FBs unless they die; in case of proliferating cells, the fluorescence intensity reduces by half at any cell division. in cultured 293 cells, I could follow up to 5-6 division (at the FACS). Carboxylate-modified FBs are stable afte fixation with PFA/FA
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining