Gentlemacs device

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Adipose tissue mononuclear cells (ATMCs) were isolated from intra-abdominal fat samples, which were first dissected then dissociated into a single cell suspension using a GentleMACS device (Miltenyi). Samples were digested in 2 ml RPMI medium (Gibco) supplemented with 20 U/ml CLSPA (Worthington) for every 4 g fat, over 1 h at 37 °C. The resulting cell suspension was washed in RPMI, and the fat layer that formed after centrifugation was decanted before the cell pellet was resuspended and filtered through a 40-µm nylon mesh. The cell suspension was then separated by Ficoll density gradient centrifugation. Isolated PBMCs and ATMCs were stored in cryopreservation medium at 10⁶ cells/100 µl [70% FCS (Gibco), 20% RPMI, 10% DMSO] in liquid nitrogen.

Tissues were digested using the Mouse Tumor Dissociation Kit (Miltenyi) on the gentleMACS device (Miltenyi) according to the manufacturer’s instructions. Red blood cell lysis buffer (BioLegend) was used to remove red blood cells. After washing with PBS, cells were incubated with TruStain fcX anti-mouse CD16/32 receptor blocking agent (BioLegend) diluted in Cell Staining Buffer (BioLegend) for 20 min at 4 °C. After washing, Zombie NIR cell viability dye (1:2,000, BioLegend) was added and incubated for 20 min at 4 °C. To assess immune cell composition, the following antibodies were added for 30 min at 4 °C: for lymphocytes, anti-CD45 PerCP Cy5.5 (30-F11, 1:100), anti-CD3 FITC (17A2, 1:100), anti-CD4 PB (RM4-5, 1:100), anti-CD8a BV 510 (53-6.7, 1:100) and anti-granzyme B AF 647 (GB11, 1:100); for macrophages, anti-CD45 PerCP Cy5.5 (30-F11, 1:100), anti-CD11B PB (M1/70, 1:100), anti-CD11C AF 488 (N418, 1:100), anti-Ly6C AF 647 (HK1.4, 1:100) and anti-Ly6G PE (1A8, 1:100), all from BioLegend. Granzyme B was added after surface staining was completed and after fixation–permeabilization (Fixation Buffer, BioLegend; 10× Intracellular Staining Perm Wash Buffer, BioLegend). Subsequently, samples were washed twice before data acquisition on the BD Aria III flow cytometer. The gating strategy is shown in Extended Data Fig. 3b.

Tumor tissue obtained by biopsy during rectoscopy or by surgical removal were transferred to 10 cm plate and cut into ≤5 mm pieces in sterile medium (RPMI 1640, 20% fetal calf serum, 2% Pen-Strep solution). Tumor pieces were dissociated into single cells using GentleMacs™ device (Miltenyi Biotec) in the presence of Collagenase I and Dispase II, followed by 40 min incubation in 37°C. cells were centrifuged and pellet was resuspended in red blood cell lysis buffer for 10 min. Washed cells were passed through 40 μM cell strained and washed with Phosphate-buffered saline, centrifuged and resuspended with flow stain buffer. Subsequently, cells were ready for immunostaining by flow cytometry.

Third-instar larvae were collected by flushing the side of their rearing tubes. The larvae were then ground using the GentleMACS™ device (Miltenyi Biotec, Bergisch Gladbach, Germany) and filtered through a series of sieves. The resulting material was loaded in 10% Ficoll solution (w/v) on top of a 15:20:25% gradient. After centrifugation, the 15:20% interface containing the enriched wing imaginal discs was collected and rehydrated in Ringer 1X (Supplementary File S1). After dissociation, filtration on 40 µm retained the cells of the salivary glands and allowed the recovery of only imaginal disc cells.

Tumor lysates were prepared in RIPA buffer (ThermoFisher) containing a 10% protease inhibitor cocktail (ThermoFisher) using a gentleMACS device (Miltenyi Biotech) based on the manufacture’s protocol. For ELISA detection, anti-hinge rabbit polyclonal antibodies (2 μg/ml) were coated on high-binding 96-well plates to capture scIgGs in tumor extracts; and anti-mouse specific antibody at 1:5000 dilution (Jackson Immune Research Laboratory, PA) with HRP conjugation was used for detection. For Western blotting detection, protein A magnetic beads were incubated with cell lysates at 4 °C for 1 hour to capture total IgGs including both intact IgGs and scIgGs, and the beads were collected and incubated with SDS containing sample buffer (Bio-Rad) at 95 °C for 5 min. The eluted samples were subjected to SDS-PAGE separation and WB detection using a goat anti-mouse IgG-Fc-HRP conjugate (1:4000) (Jackson Immune Research Laboratory, PA) as previously described^{10 (link),12 (link)}.

Spleens were squeezed through 100 µm cell strainers and washed with PBS (300xg, 10 min, 4°C) followed by erythrocytes lysis using hypo-osmolaric NaCl buffers (0.2% NaCl, 1.6% NaCl). Lysis was stopped by filling up with PBS and centrifugation by 300xg, 10 min and 4°C. The supernatant was discarded and the pellet was resuspended in sterile PBS and splenocytes were passed through a 30°µm cell strainer and stored on ice until further analysis: Liver cells were separated using the gentleMACS device (Miltenyi Biotec) and the Liver Dissociation Kit (Miltenyi Biotec) according to manufacturer recommendations. Afterwards erythrocytes lysis was performed by hypo-osmolaric NaCl buffers as described for splenocytes. Cells were passed through a 30 µm cell strainer and a density gradient centrifugation was performed with Percoll (GE Healthcare, ϱ=1.041 g/mL, 360xg, 20 min, RT). Cells were washed twice with PBS and resuspended in PBS and stored on ice until further analysis.