Multiparameter Flow Cytometry Analysis of Cellular Activation

5x10E4 to 1x10E5 cells were labelled for 30 minutes in 18 µl to 100 µl PBS + 1% BSA containing one or combinations of the following antibodies: anti-CD16-PECy7 (Biolegend; 1:1000; 3G8), anti-CD62 L-APCCy7 (Biolegend; 1:25; Greg-56), anti-CD63-APC (Biolegend; 1:100; H5 C6), anti-CD66b-PE (Biolegend; 1:100; G10F5), DAPI (Sigma-Aldrich), anti-CD3-PacificBlue (Beckman Coulter, 1: 50, UCHT-1), anti-CD14-FITC (Miltenyi, 1:100, Tük 4), anti-ICAM-1-AlexaFluor-405 (SantaCruz, 1:100, sc-107-af405), an in-house VHH against P-selectin (1:125, B10.6 [43 (link)],), or Annexin-V (250 ug/ml, VPS diagnostics, cat. nr. A705). Neutrophil activation was assessed by measuring the increase in CD62L expression [44 (link)] and the simultaneous decrease in CD16 and increase in CD63 expression [45 (link),46 (link)]; HUVEC activation was assessed by evaluating the increase in ICAM-1 expression [47 (link)]; red blood cell activation was assessed by evaluating phosphatidylserine exposure detected using annexin-V staining [48 (link)]; platelet activation was assessed by investigating P-selectin expression [49 (link)]. Surface labelling of cells was measured on a FACSCanto (BD Biosciences) or a LSR-II (BD Biosciences) flow cytometer relative to unlabelled controls.

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Driedonks T.A., Mol S., de Bruin S., Peters A.L., Zhang X., Lindenbergh M.F., Beuger B.M., van Stalborch A.M., Spaan T., de Jong E.C., van der Vries E., Margadant C., van Bruggen R., Vlaar A.P., Groot Kormelink T, & Nolte-‘T Hoen E.N. (2020). Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases. Journal of Extracellular Vesicles, 9(1), 1764213.

Publication 2020

anti-CD16-PECy7 anti-CD62 L-APCCy7 anti-CD63-APC anti-CD66b-PE DAPI anti-CD3-PacificBlue anti-CD14-FITC FACSCanto

Annexin v Anti cd3 Antibodies Cd62l Cd66b Cells Dapi Diagnostics Fitc Icam 1 Neutrophil activation P selectin Phosphatidylserine Platelet activation Red blood cell

Corresponding Organization : Utrecht University

Other organizations : Amsterdam University Medical Centers, University of Amsterdam, University Medical Center Utrecht, Sanquin

Top 5 similar protocols

Variable analysis

independent variables

Concentration of PBS + 1% BSA (18 μl to 100 μl)
Combinations of the following antibodies:
Anti-CD16-PECy7 (Biolegend; 1:1000; 3G8)
Anti-CD62 L-APCCy7 (Biolegend; 1:25; Greg-56)
Anti-CD63-APC (Biolegend; 1:100; H5 C6)
Anti-CD66b-PE (Biolegend; 1:100; G10F5)
DAPI (Sigma-Aldrich)
Anti-CD3-PacificBlue (Beckman Coulter, 1: 50, UCHT-1)
Anti-CD14-FITC (Miltenyi, 1:100, Tük 4)
Anti-ICAM-1-AlexaFluor-405 (SantaCruz, 1:100, sc-107-af405)
An in-house VHH against P-selectin (1:125, B10.6 [43 (link)])
Annexin-V (250 ug/ml, VPS diagnostics, cat. nr. A705)

dependent variables

Increase in CD62L expression (neutrophil activation)
Decrease in CD16 and increase in CD63 expression (neutrophil activation)
Increase in ICAM-1 expression (HUVEC activation)
Phosphatidylserine exposure detected using annexin-V staining (red blood cell activation)
P-selectin expression (platelet activation)

control variables

Labelling duration (30 minutes)
Cell concentration (5x10^4 to 1x10^5 cells)

positive controls

Unlabelled controls

negative controls

Not explicitly mentioned

Annotations

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