hello
i collect MSC and i want to do immunofluorescene to detect the surface antigen CD -73 AND CD 90 , it is the first time i want to do flow cytometer .
someone has a video or a protocol detailed in which explain how can i stain cell surface marker for MSC to make then analysis
Hi Linda,
if it is the first time you do some flow cytometry you need some basic information to choose the right strategy setting up your experiment. A nice Tutorial Video is on this website:
http://www.miltenyi.tv/#top
Just click on Tutorial and go to page 5. There you find a video called Multicolor Flow Cytometry. I hope this helps.
Greetings, Julia
I suggest you to read papers of Zuba Surma E. teams and Ratajczak MZ team.
they did pretty intensive studies on MSC and their analysis with Flow Cytometer.
If this is your time with flow cytometry, it is always best to do some background reading on the principle behind the procedure. As for the main protocol, the basic steps for staining with cell surface makers (for ex. CD73, CD90) include:
cell trypsinization (if adherent cells) --> wash with 1x PBS --> repeat wash --> count cells --> aliquote ~1 mil cells in each tube (for ex. atleast 4 if using 2 markers) --> wash again with 1x PBS --> stain Ab with staining buffer IN DARK --> incubate as given in Ab literature --> wash with 1x PBS --> immediately acquire or add fixation buffer and store in 4 deg.
Important points here include:
1. If you are using more than one Ab per acquisition, always keep a single-stained tube of each Ab apart from the dual/ triple stained tubes as it serves as compensation control.
2. Always maintain cold chain while processing the cells, like using cold PBS for washes, maintaining the centrifuge before start of the experiment.
3. It is always economical if you titrate your Ab before starting with major experiments; it saves a lot of Ab per experiment as it generally turns out that a much lower volume than what is given in company literatures suffices for the given no. of cells.
4. The no. of aliquotes you require would depend on whether you wish add extra control to your experiment, like isotype controls and FMO control (do intensive background reading to see their role in an experiment).
Hope this was helpful.
Thank you very much all my friend ! All these nformations are helpful
@francesco gubinelli please could you send me the link of these papers to read it thx in advance
MSC's flow cytometry is very tricky. For first runs I would recommend to use one marker at a time for staining as MSCs are highly autofluorescent (don't put too many markers/colors altogether), and always have unstained controls of cells grown in SIMILAR conditions . if you add nutrients MSCs grow in size and change cells structure and it will affect forward/side scatter characteristics, so "starving" cells compare to cells grown in more rich in nutrients medium (also more confluent) will have different background autofluorescence. If you use one antibody at a time you don't need compensation and Fluorescent minus one controls =FMOs.
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