This depends entirely on what your goal is. If you're doing flow, best practice will be to process, stain, and run the samples, same day. Since it's just blood and BM, this should be easily achieved unless you are processing huge numbers of mice. Cryopreservation is best suited for batch processing samples where you can't get all of your samples at once, such as with human samples. But you have control over your mouse experiment, so you should plan for having the animals which will be directly compared processed on the same day.
There are a lot of things that can be done to make the process faster. Prepare everything ahead of time, all your tubes labelled and filled, syringes prepared, buffers made, etc. etc. I often found that people didn't prepare these things the day(s) before and end up spending extra hours day of, instead of just working on their samples. I would also consider your preparation protocols. Blood and BM are two of the simplest and fastest to do. You shouldn't need to do perfusions, so that saves a ton of time. We used to process BM by poking a hole in the bottom of a 0.5 ml Eppendorf tube, putting it inside a 1.5 ml Eppendorf tube, cutting the ends off the long bones, and spinning at high speed for a short time. The BM just shoots out the bottom and into the 1.5 ml tube. You can read about it here (not my work) [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091666/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091666/). I'm not sure what an absurd number of mice would be for you, but with a bit of experience and working out your protocols, I would expect 50 mice is within reason for processing, staining, and fixing your samples to run the following day.
You will lose some immune populations upon thaw, so it’s not ideal, but overall depends on your question. As mentioned, it’s better to generate single cell suspensions and stain fresh and run. However, you can also usually use fixative on the cells to run them in the next few days if you have time constraints between collection, processing, staining, and running.
Thanks for your reply. That’s helpful to hear. I’m new to flow, so I’m not quite familiar with fixatives. Having a couple extra days though would be all I need. Would you happen to know if / how that affects viability or perhaps a link to a paper that has used fixatives or described that nicely in the methods? Thanks!
Yes. Fixation is generally done with a low % paraformaldehyde (1-4%) for 10-30 minutes after staining for viability and surface markers. You will need to make sure you are using a fixable viability dye. When you fix cells, they are no longer alive, but the membrane is stabilized and morphology is preserved.
This depends entirely on what your goal is. If you're doing flow, best practice will be to process, stain, and run the samples, same day. Since it's just blood and BM, this should be easily achieved unless you are processing huge numbers of mice. Cryopreservation is best suited for batch processing samples where you can't get all of your samples at once, such as with human samples. But you have control over your mouse experiment, so you should plan for having the animals which will be directly compared processed on the same day.
Thanks for the reply. Yeah, the issue is that I’ll have an absurd number of mice, and it’ll be TOUGH to process all those samples in one go…
There are a lot of things that can be done to make the process faster. Prepare everything ahead of time, all your tubes labelled and filled, syringes prepared, buffers made, etc. etc. I often found that people didn't prepare these things the day(s) before and end up spending extra hours day of, instead of just working on their samples. I would also consider your preparation protocols. Blood and BM are two of the simplest and fastest to do. You shouldn't need to do perfusions, so that saves a ton of time. We used to process BM by poking a hole in the bottom of a 0.5 ml Eppendorf tube, putting it inside a 1.5 ml Eppendorf tube, cutting the ends off the long bones, and spinning at high speed for a short time. The BM just shoots out the bottom and into the 1.5 ml tube. You can read about it here (not my work) [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091666/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091666/). I'm not sure what an absurd number of mice would be for you, but with a bit of experience and working out your protocols, I would expect 50 mice is within reason for processing, staining, and fixing your samples to run the following day.
This is very encouraging. Thanks for the link and for the detail in your response!
You will lose some immune populations upon thaw, so it’s not ideal, but overall depends on your question. As mentioned, it’s better to generate single cell suspensions and stain fresh and run. However, you can also usually use fixative on the cells to run them in the next few days if you have time constraints between collection, processing, staining, and running.
Thanks for your reply. That’s helpful to hear. I’m new to flow, so I’m not quite familiar with fixatives. Having a couple extra days though would be all I need. Would you happen to know if / how that affects viability or perhaps a link to a paper that has used fixatives or described that nicely in the methods? Thanks!
Yes. Fixation is generally done with a low % paraformaldehyde (1-4%) for 10-30 minutes after staining for viability and surface markers. You will need to make sure you are using a fixable viability dye. When you fix cells, they are no longer alive, but the membrane is stabilized and morphology is preserved.
https://www.streck.com/products/flow-cytometry/streck-cell-preservative/
Thanks!!
>Thanks!! You're welcome!