Acidic Tumor Spheroid Labeling

3D tumor spheroids were incubated for 24 h with 2 µM Alexa 568-conjugated pH-low insertion peptide variant 3 (pHLIP V3; NH₂-ACDDQNPWRAYLDLLFPTDTLLLDLLW-COOH) (Weerakkody et al, 2013 (link)) to label acidic regions; an acid-independent Alexa 594 K-pHLIP peptide wherein aspartate residues were replaced by positively charged lysine residues was used as negative control. After cell dissociation using Accutase, cells were resuspended into PBS for FACS sorting. FACSAria III cell sorter (BD Biosciences) was used to isolate Alexa 568 pHLIP-positive and Alexa 594 K-pHLIP-negative cells.

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Aubert L., Bastien E., Renoult O., Guilbaud C., Özkan K., Brusa D., Bouzin C., Richiardone E., Richard C., Boidot R., Léonard D., Corbet C, & Feron O. (2024). Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors. EMBO Reports, 25(3), 1469-1489.

Publication 2024

FACSAria III cell sorter

Corresponding Organization : Walloon Excellence in Lifesciences and Biotechnology

Other organizations : Directorate-General Joint Research Centre, Centre Georges François Leclerc, Cliniques Universitaires Saint-Luc

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Variable analysis

independent variables

Incubation of 3D tumor spheroids with 2 μM Alexa 568-conjugated pH-low insertion peptide variant 3 (pHLIP V3; NH2-ACDDQNPWRAYLDLLFPTDTLLLDLLW-COOH) for 24 h

dependent variables

Isolation of Alexa 568 pHLIP-positive and Alexa 594 K-pHLIP-negative cells using FACS sorting

control variables

Incubation of 3D tumor spheroids with an acid-independent Alexa 594 K-pHLIP peptide wherein aspartate residues were replaced by positively charged lysine residues as a negative control

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