Hi all,
I would have to irradiate lymphoma cell lines (Bl41 and J3D) and planning on looking for DNA damage clearance by IF following different nuclear markers. The center where I will irradiate the cells has no cytospin and so for short time point I would not be able to get the cells to my lab on time.
One solution would be to fix the cells before cytospin but I cannot find any protocol for that. Has anybody try to fix floating cells before cytospin?
Thanks,
Matt
you have to invent a new technique...cytospin is to let cells on a coverslip or slide to be stained or labeled...
Which is exactly what I want to do, staining for nuclear markers. The issue is being able to attach the cells after fixation and not before as I would not be able to cytospin the cells before fixing them.
Have a look at thin-prep preperation or liquid cytology. This technic is used instead of gyn-smears. Cells are brought into a fixing solution and slides are produced afterwards.
Other option is to make a cellblock in paraffin and get paraffinslides.
Thanks, I will check that, I also found a protocol on RnD website that seems okay for what I want to do...https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-non-adherent-cells
If cytospin after fixation is not possible:
You can do IF-taining in tube (1.Antibody, washing steps 2.Antibody, DNA-staining ...)
and fix the cell before microscopy on the slide with low melt 0.5%-1% agarose.
No issue at all, I developed techniques over 20 years ago. Simply re-suspend your cells in medium and add formaldehyde (1:10 producing a 4% solution of formalin). I recommend using 'charged' slides for your cytospins and I spin at 500rpm for 10 minutes. The remaining cell suspension can quite happily be stored in the fridge for 12 months+ and more cytospins made at any point. I've used this for HCC, breast cancer, prostate cancer cell lines to name but a few. I've attached images of EBV+ lymphoma cell lines to demonstrate the results.
Formalin fixation obviously will then require some form of antigen retrieval technique, pre-immunostaining. Having been at the forefront in developing these in the UK in the early 90's I actually published on the use of microwave antigen retrieval for cytological preparations (Reynolds et al Cytopathology 1994; 5: 345-358).
Some say this is problematic for fluorescent techniques, but that is a myth. I use pretty much the same protocols for light or fluorescent microscopy, particularly on formalin fixed paraffin embedded tissues, as evident by my numerous publications showing this, eg Reynolds et al Hepatology. 2008;47(2):418-27.
Hope this resolves your problem and please feel free to contact me direct if you experience any specific issues.
Regards
Gary
Hi Gary,
thanks so much for all those details. I will try that ASAP and let you know if I need more details. Thanks
Matt
- Badges
- Science method
- Similar topics
- Biological Science
- General Biochemistry
- Biochemical Techniques
- Cytology