Apoptosis Evaluation by Flow Cytometry

Apoptosis was evaluated by flow cytometry (BD FACSCanto) using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™), following the instructions provided by the manufacturer. Briefly, differentiated CAD cells (48 h) were treated with the ACM media or Etoposide 10 µM for 24 h [50 (link)], and then harvested by trypsinization for 1 min at 37 °C. Following two rounds of washes with PBS, cells were resuspended in the binding buffer and incubated with PI and Annexin V-Alexa Fluor 488 for 30 min. Data were processed using the FlowJo v10.8.0 Software [51 (link)].

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Palacios E., Lobos-González L., Guerrero S., Kogan M.J., Shao B., Heinecke J.W., Quest A.F., Leyton L, & Valenzuela-Valderrama M. (2023). Helicobacter pylori outer membrane vesicles induce astrocyte reactivity through nuclear factor-κappa B activation and cause neuronal damage in vivo in a murine model. Journal of Neuroinflammation, 20, 66.

Publication 2023

BD FACSCanto FITC Annexin V Apoptosis Detection Kit I

Alexa fluor 488 Annexin i Annexin v Apoptosis Buffer Cells Etoposide Fitc Flow cytometry

Corresponding Organization :

Other organizations : Universidad Central de Chile, Advanced Center for Chronic Diseases, University of Chile, University of Washington

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Variable analysis

independent variables

Treatment with ACM media
Treatment with Etoposide 10 µM

dependent variables

Apoptosis evaluated by flow cytometry using FITC Annexin V Apoptosis Detection Kit I

control variables

Differentiated CAD cells (48 h)
Harvesting of cells by trypsinization for 1 min at 37 °C
Two rounds of washes with PBS
Resuspension of cells in binding buffer
Incubation with PI and Annexin V-Alexa Fluor 488 for 30 min

controls

Positive control: Etoposide 10 µM treatment
Negative control: ACM media treatment

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