PB0893 - Granulocyte isolation by double and single ficoll density gradient centrifugation: comparison of cell yield, phosphatidylserine expression and oxidative burst reactivity

Background: Platelet-neutrophil interactions are widely studied in the field of thromboinflammation. In vitro studies usually use density gradient centrifugation methods for granulocyte isolation which lead to cell activation. Isolation-induced priming may complicate interpretation of experimental results.

Aims:

Aim: To compare various methods of granulocyte isolation from peripheral blood by cell yield, phosphatidylserine expression and oxidative burst reactivity.

Methods: Granulocytes were isolated from citrate-stabilized peripheral blood of healthy volunteers by single or double ficoll density gradient centrifugation. In case of single density gradient centrifugation preliminary or subsequent dextran sedimentation of erythrocytes was performed. Obtained granulocyte suspensions were investigated by flow cytometry. Statistical analysis was performed using One-Way ANOVA with post-hock Tukey test.
All experiments were performed in accordance with Helsinki Declaration.
This work is supported by grant B23-RSF162 and grant 23-45-10039.

Results: Isolation by double density gradient yielded more granulocytes (per ml of blood) than isolation by single density gradient (fig.1). Yield of granulocytes was reduced if erythrocytes were sedimented before single gradient centrifugation (fig. 1). Percent of granulocytes in resulting suspension equally high at isolation by double density gradient centrifugation and single density gradient centrifugation with subsequent dextran sedimentation of erythrocytes and significantly reduced at isolation by single gradient centrifugation with preliminary erythrocyte sedimentation. For all methods content of basophils and eosinophils in resulting suspension was equal and didn’t exceed 0,2 and 3% respectively. Isolation by single gradient led to higher amount of AnnexinV positive granulocytes, which significantly increased when erythrocytes were sedimented before gradient centrifugation (fig. 2). Oxidative burst of granulocytes in response to fMLP was higher when a single gradient isolation was used.

Conclusion(s):

Conclusion: Obtained results indicate that isolation of granulocytes by double ficoll density gradient centrifugation is optimal for in vitro studies as it lead to the highest yield and has less impact on cell functions.