Comparison of Three Isolation Techniques for Human Peripheral Blood Mononuclear Cells: Cell Recovery and Viability, Population Composition, and Cell Functionality.
Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments, these three PBMC isolation techniques were compared for cell recovery and viability, PBMC population composition, and cell functionality, aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors, applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures, with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability, assessed by trypan blue exclusion, was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures, for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities, these data may support selection of a specific PBMC isolation technique for downstream analysis.
1 Good Biomarker Sciences , Leiden, the Netherlands .
Hendrika W
HW
Grievink
1 Good Biomarker Sciences , Leiden, the Netherlands .|2 Centre for Human Drug Research , Leiden, the Netherlands .
Tarik
T
Luisman
1 Good Biomarker Sciences , Leiden, the Netherlands .
Cornelis
C
Kluft
2 Centre for Human Drug Research , Leiden, the Netherlands .
Matthijs
M
Moerland
1 Good Biomarker Sciences , Leiden, the Netherlands .
Karen E
KE
Malone
D049109:Cell Proliferation; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D002470:Cell Survival; D016207:Cytokines / Q000378:metabolism; D064368:Healthy Volunteers; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000378:metabolism; D008297:Male
D016207:Cytokines; D007770:L-Lactate Dehydrogenase
cell viability; cytokines; density centrifugation; peripheral blood mononuclear cells (PBMCs)