Hi everybody,
I want to detect CD4/CD8 T lymphocyte in the mouse whole blood samples by flow cytometry. Because the flow cytometry is not ready in my lab and I need to collect the samples, I want to store the sample for 3 days. What should I do? I think about 2 options: (1) store the whole blood samples until I'm ready to analyse, (2) stain the sample with labelled antibody, fix the cell with PFA 1%, after that store the sample.
Hope your help!
Many thanks!
Nuong
Hi,
You could fix the cells in 2% PFA and store them for a while. Storing stained cells could lead to loss of fluorescent signal over time. I have fixed cells and stained them for CD4/CD8 unto a month later with no adverse effects noticed.
Best,
Deepika.
Most Immunology procedures- should decsribe this!
For a start (i) use CPDA-1 as the anticoagulant-available from suppliers ready correct vol in bags (67ml in the bag (BAXTERS), VACUTAINER (BD) (advantage- siliconised, prevent cells sticking) AND SARSTEDT, Germany: Monovetttes- syringe -tubes-(~8ml and ~6ml capacities)- after taking the blood- the plunger top snaps off at the base of the handle', leaving you with a tube to transport-
(ii) For harvesting lymphocytes, neutrophils, monocytes- by Histopaque 1077, Ficoll-Met, PERCOLL,- REMOVE PLATELETS -TROUBLE- by spinning blood (15 min ~1000 rpm at room temp (~22˚C) (unfortunately- I don't think- Monovettes are said to be suitable for spinning (supplier instructions)-
Should you wish to know more- please feel free to write:
Hey,
i would also advise you to perfom a Ficoll density gradient centrifugation first, fixate your cells, store them and stain them only shortly before acquirement. This way you should have the least adverse effects. If you want to stain them prior to fixation and storage, make sure to keep your samples dark all the time and have some proper compensation controls.
Bes,
Sarah
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