Isotype matched igg1 control antibody

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.

The expression of memHsp70 on tumor cell lines was determined by flow cytometry using either the FITC-conjugated cmHsp70.1 antibody or the CF-labeled 14-mer peptide TPP, both of which bind to the exposed sequence of memHsp70. Briefly, after incubation of viable cells (2×10⁵ cells) with the antibody or peptide for 30 min at 4°C and following two washing steps, viable (propidium iodide negative) cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, U.S.A.). An isotype-matched (IgG1) control antibody (BD Biosciences) was used to evaluate non-specific binding to cells.

In vitro binding of the DOTA-αGPC3 conjugate was evaluated by flow cytometry. HepG2-Red-FLuc cells were grown as above until 70% confluent and then detached with 0.25% trypsin, counted, washed, and resuspended in cold phosphate-buffered saline (PBS) at a concentration of 1 × 10⁶ cells/mL. One microgram of unconjugated or DOTA-αGPC3 primary antibody was added to the cell suspension and incubated for 45 minutes on ice. Primary antibody control samples received 1 μg of isotype-matched IgG1 control antibody (BD Biosciences, cat. no. 555746). Unstained samples did not receive primary antibody. The cells were then washed in cold PBS, and 1 μg of FITC-labeled goat-α-mouse IgG1 secondary antibody (Southern Biotech, cat. no. 1070-02) was added to the cell suspension and incubated on ice for 30 minutes in the dark. Unstained samples did not receive secondary antibody. The cells were washed in cold PBS, fixed with 1% paraformaldehyde for 15 minutes, and then washed and resuspended in cold PBS. Fixed cells were analyzed with a BD FACSCanto flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ) using the FACSDiva software. A minimum of 10,000 cells were analyzed for each sample in triplicate. Data analysis was performed on the FlowJo software, version 8.8.6 (Tree Star, Ashland, OR).