Hsp70 Expression in Human Organoids

To confirm the expression of membrane-associated Hsp70 in malignant cells of the human organoids, flow cytometry was performed on single cell suspensions from human organoids as follows. Untreated, viable organoids (passage 3–6) were washed with PBS plus 10% FBS and subsequently disassembled by incubation in EDTA-trypsin (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 0.5U/ml collagenase (Roche Diagnostics, Mannheim, Germany) for 10 min at 37 °C. After enzyme deactivation with PBS plus 10% FBS, the single cell suspension from organoids was centrifuged again and the pellet was incubated with isotype-matched (IgG1) control antibody (BD Biosciences) or FITC-conjugated cmHsp70.1 antibody for 30 min at 4 °C protected from the light as described previously [43 (link)]⁠. Only viable cells (propidium-iodine negative) were then analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry data were then quantified by using the mean fluorescence intensity and/or the percentage of Hsp70-positive cells, obtained after subtracting the contribution from cells bound to the IgG1 control antibody.