Talk:WG4 MitoEAGLE data: blood and cultured cells

Respiratory protocols PBMCs

Elisa Calabria 2016-10-31

I’m still working with PBMCs and mitochondrial function. I’d like to receive your impressions on the main points on which we could focus in the discussions in Verona, to achieve an agreement on procedures, protocols, crucial steps or any other point we would like to fix as major starting point. I know that we’re working in different labs and it is difficult to have a continuous feedback. But despite of our expertise I guess that we still have many open points. For example in the last period I started a project on aging and decided to stick more to the “Innsbruck” protocol. I note that using the Luecosep tubes versus only Ficoll gradient (I was doing that previously) the consistence of the PBMCs layer is rather different, more packed with all the platelets. But I also think that we can’t loose ourself in too many details, we have to give some major points of agreement.

So if you agree I’ll try to summarize the major points of our procedure, set them into the context of MITOEAGLE, and open a discussion/dialogue, if you think this could be useful.

Major points:

Blood collection fasten/feeded - time of the day
First dilution steps (PBS/RPMI- I guess we decided for the PBS, I was using the RPMI and worked very well, so maybe it’s not so crucial)
Leucpsep tubes and 1st centrifugation (here I think also we already agreed for the shorter leucosep tubes centrifugation)
Number of cells (at least 1*10⁶) - or cell density (1*10⁶/ml)
Cryopreservation - I’ll come back on this in Verona
SUIT protocol: here I think it’s still open, I know that Zuzana was setting a long and complete protocol, I don’t know if all of us are using this version, I had the impression it was very long. But I’d like to receive your versions/opinions.

Please, I’d like you to consider this mail not as an investigation, but just an attempt to open the dialogue in view of the Verona meeting. Sorry if I didn’t take this time before.

Best wishes - Elisa