Neutrophil Apoptosis Assay Protocol

CD10^neg/CD10^pos neutrophils (1x10⁶/mL) were cultured for 24 hr in RPMI 1640 medium supplemented with 5% HI-FCS and 1% PenStrep. Apoptosis rate was assessed by flow cytometry using a Vybrant DyeCycle Violet/SYTOX AADvanced Apoptosis Kit (A35135, Invitrogen) and Apotracker Green (Barth et al., 2020 (link)) (427402; Biolegend), according to the manufacturer’s protocols. Stained neutrophils were analyzed using a Gallios cytometer and Gallios software (Beckman Coulter).

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Fraccarollo D., Neuser J., Möller J., Riehle C., Galuppo P, & Bauersachs J. (2021). Expansion of CD10neg neutrophils and CD14+HLA-DRneg/low monocytes driving proinflammatory responses in patients with acute myocardial infarction. eLife, 10, e66808.

Publication 2021

Vybrant DyeCycle Violet/SYTOX AADvanced Apoptosis Kit Apotracker Green Gallios cytometer Gallios software

Apoptosis Dyecycle violet Flow cytometry Neutrophils

Corresponding Organization :

Other organizations : Medizinische Hochschule Hannover

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Variable analysis

independent variables

Culture duration (24 hr)

dependent variables

Apoptosis rate

control variables

Cell type (CD10^neg/CD10^pos neutrophils)
Cell density (1x10^6/mL)
Culture medium (RPMI 1640 with 5% HI-FCS and 1% PenStrep)
Apoptosis assay (Vybrant DyeCycle Violet/SYTOX AADvanced and Apotracker Green)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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