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Talk:WG4 MitoEAGLE data: blood and cultured cells

From Bioblast

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:
  1. Blood collection fasten/feeded - time of the day
  2. 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)
  3. Leucpsep tubes and 1st centrifugation (here I think also we already agreed for the shorter leucosep tubes centrifugation)
  4. Number of cells (at least 1*106) - or cell density (1*106/ml)
  5. Cryopreservation - I’ll come back on this in Verona
  6. 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

O2k-Protocol to be updated

  • Sumbalova Z, Droescher S, Hiller E, Chang S, Garcia L, Calabria E, Volani C, Krumschnabel G, Gnaiger E (2016) Isolation of blood cells for HRR. Mitochondr Physiol Network 21.17(02):1-15. - »Bioblast link«
A blood sample of 18 ml is presently required for four O2k chambers with PBMCs (2-3 million cells per ml) and four to six O2k chambers with platelets (100-125 million cells per ml).

Nina Krako 2016-10-31

I need to have an idea on how much blood is required to obtain a relevant basic measurement on high-resolution respirometry with PBMCs. I know that we will harmonize these procedures along with MitoEAGLE project, but at the moment I would need to have a rough idea of number of cells needed, because of planning some future studies.
PBMC isolation protocol
  1. About 18 ml of human blood from vacutainer transfer to 50 ml Falcon tube.
  2. Centrifuge at 300xg, 10 min, accel 5, decel slow, 22˙C
  3. Collect plasma with ~2 mL erythrocytes (around 10 mL all together) and pour onto 5 mL Limfoprep (Fikol), in 15 mL Falcon tube
  4. Centrifuge 1000xg 20 min, accel 5, decel off, 22˙C (last about 40 min)
  5. Collect entire ring of cells and add up to 15 mL PBS or cell medium (RPMI) (depending on the purpose: cell lysis or culturing)
  6. Centrifuge 500xg, 10 min, break on max, 22˙C
  7. Resuspend cells into 15 mL RPMI
  8. Centrifuge 500xg 10 min, break on max, 22˙C
  9. Resuspend cells with 2 mL RPMI
  10. Count cells
1 mL of blood yields about 0,5 - 1,0 million of cells (depends on subject/patient).
For FACS analysis we use 200.000 – 250.000 cells per well (24-well plate).
For RNK isolation (5 million of cells).
The rest of cells are lysed for WB analysis (5-12 million).