Calabria 2017 MiP2017 WG4

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Calabria Elisa
PBMCS purification and mitochondrial function assay: monitoring preparative steps and comparison of protocols.

Link: MiP2017

Calabria E, de Jong V, Tarperi C, Schena F (2017)

Event: MiP2017

COST Action MITOEAGLE

In our laboratory we are mainly focused on PBMCs blood cells. One of the main issues of working with PBMCs is the quality of the final batch of cells. In particular the contamination of platelets in the final sample could affect the respiratory profile of the sample when evaluating mitochondrial function. We have monitored all the steps of the PBMCs purification procedure with a Sysmex XN-1000 hematology analyzer. This instruments allows a high degree of accuracy of platelets counts, also using a specific fluorescent reagent [1]. The data collected show that we achieve 97% depletion of platelets after two washing steps in RPMI, and we collect the 30% of the initial amount of PBMCs.

One of the main aim of WG4 is also protocols in the harmonization to SUIT reference protocols (RP1 and RP2). Since we are not currently running exactly the suggested RP protocols, we tried to verify if at least some common functional steps of the protocol are comparable to RP1 and RP2 protocols. Preliminary data show that at least in some common steps there is no significant difference between the old protocol and the suggested RP1 and protocol in PBMCs human cells, further experiments are needed to evaluate RP2.


Bioblast editor: Beno M, Kandolf G O2k-Network Lab: IT Verona Calabria E


Labels:


Tissue;cell: Blood cells 





References

  1. Wada A, Takagi Y, Kono M, Morikawa T (2015) Accuracy of a new platelet count system (PLT-F)depends on the staining property of its reagents. PLOS ONE 10:1-13.