Gentlemacs machine

Hamsters were anesthetized by intraperitoneal injection of a mixture of 100 mg kg⁻¹ of ketamine/5 mg kg⁻¹ of xylazine. After loss of toe pinch reflex, SARS‐CoV‐2 was administered to each hamster via intranasal route in a total volume of 50 µL. Animals were then administered reversal agent (atipamezole, 0.15 mg kg⁻¹) and placed on a heating pad until able to right themselves. Body weights and clinical signs were checked and recorded daily. For sample collection, hamsters were anesthetized as described above and administered pentobarbital (100 mg kg⁻¹) via intraperitoneal injection. After exsanguination and pneumothorax, tissues were collected aseptically for analyses.
Whole lungs from individual hamsters were placed in 2 mL of DMEM 1% FBS containing antibiotics/antimycotics (D1) in C tubes and homogenized using a GentleMACS machine at “lung 2” setting (Miltenyi). After centrifugation for 10 min at 1000 × g, supernatant was removed, and remaining homogenates were resuspended in Trizol for RNA extraction. Chloroform‐based phase separation and RNA precipitation were then performed followed by two ethanol washes.

Mice were sacrificed and lung and/or MLN tissues were harvested at the indicated times post-infection. In most cases, tissues from 3–7 mice were pooled to generate one sample. Lung tissues were disrupted by tissue dissociation using a GentleMACS machine (Miltenyi). Mononuclear cells were purified using Fico-LITE at room temperature, washed, then resuspended in 10% RPMI. MLN tissues were ground between the ends of two frosted glass slides and mononuclear cells were isolated as described above. After isolation, cell preparations were stained with fluorochrome-labeled antibodies to the following cell surface markers: CD11c (N418), CD11b (M1/70), CD103 (2E7), PDCA (clone), CD8α (53-6.7), MHC class II (M5/114.15.2), CD3 (145-2C11) purchased from BD biosciences, eBioscience, Miltenyi or Biolegend at concentrations predetermined by antibody titration. All staining was done for 20 minutes at 4°C in FACS staining buffer (PBS + 1% FBS + 0.05% sodium azide). Samples were collected on an LSR-II flow cytometer (BD, San Jose, CA) and data were analyzed using FlowJo version 9.9.10, the gating strategies for lung and MLN are presented in Figures S1 and S2, respectively.

Mice were euthanized with an intraperitoneal injection of 2.5% Avertin, followed by removal of the lungs from the chest cavity. The lungs were then placed in 5 ml of RPMI media containing 1.3 MandlU of Liberase and 0.2 mg DNase. The tissue was dissociated using a lung-optimized program on a Miltenyi Biotec gentleMACS machine followed by a 30-minute incubation at 37°C. Using the gentleMACS dissociator, the tissue was homogenized. Red blood cells were lysed, and 2 million cells were plated for antibody staining.

For the majority of experiments the cells were processed as follows. Splenocytes were harvested from naïve TCR Tg mice and brought into a single cell suspension by tissue dissociation using a GentleMACS machine (Miltenyi). Lymphocytes were purified using Fico-LITE (Atlanta Biologicals), washed, and resuspended in lymphocyte medium (RPMI, 10%FBS, 2 mM Glutamine, 1 mM Sodium Pyruvate, non-essential amino acids, 25 mM HEPES, 5×10⁻⁵ M β-mercaptoethanol and Pen/Strep antibiotics). Lymphocytes were stimulated with peptide at concentrations ranging from 1×10⁻⁶ M to 1×10⁻¹² M, in the presence of 1 µg/ml of costimulatory antibodies for CD28 and CD49d (BD Biosciences) and 10 U/mL IL-2 (PeproTech, NJ) unless stated otherwise. The stimulation was carried out in the presence of feeder cells: 5000 rad-irradiated CB6F1 Thy1.1+ splenocytes in a ratio of 1∶1 with the stimulated cells. All stimulations were carried out in 37°C, 5% CO₂ humidified incubator for the indicated time points.

At study termination, tumour samples from the Humabody negative control group and the CB213 10 mg/kg group were collected, minced into fine pieces and transferred into C-tubes (Miltenyi) containing 2.5 ml digestion buffer (Tumour dissociation kit) and disaggregated in a Miltenyi GentleMACS machine. Cells were filtered through 70 μm mesh, centrifuged and washed twice with RPMI 1640. Cells were counted and Fc blocked (purified rat anti-mouse CD16/32 BD Fc block: BD 553141) before staining. Tumour cells were stained with fluorochrome-conjugated antibodies to mCD45, mCD4, mCD8, hPD1 (Biolegend: Cat. 103132, 100406, 100708, 329918, respectively) and to mCD3, hLAG3 (BD: Cat. 740268, 565716).

Lungs were weighed and quick-frozen in 10% MEM-10, and plaque assays were performed as described previously (62 (link)). Briefly, thawed lung tissue was dissociated using a GentleMACs Machine (Miltenyi Biotec). Cell suspensions were pelleted to remove cellular debris, and clarified supernatants were serially diluted and inoculated in triplicate on 80% confluent HEp-2 cell monolayers. After rocking for 1 h at room temperature, monolayers were overlaid with 0.75% methyl cellulose in MEM-10 and incubated at 37°C. Cells were fixed with 10% buffered formalin and stained with hematoxylin and eosin on day 4. Plaques were counted and expressed as Log₁₀ PFU/g of lung tissue. The limit of detection was 1.8 Log₁₀ PFU/g.

Mice were killed according to animal welfare guidelines; tumour tissue and blood were isolated in the animal facility. Tumour tissue was transferred to PBS and was disrupted using manual scissors and the Miltenyi Gentle MACS machine. Subsequently, it was digested in an enzyme mix consisting of RPMI with 10 mg ml^–1 DNase (Sigma-Aldrich) and 0.25 mg ml^–1 Liberase (Sigma-Aldrich).After 30 min of digestion at 37 °C, the tissue mix was filtered through a 70-µm filter and resuspended as a single-cell suspension with an appropriate volume for subsequent staining with fluorescently labelled antibodies. Blood was transferred to heparin tubes, and red blood cells were lysed with erythrocyte lysis buffer. After red blood cell lysis, cells were resuspended as a single-cell suspension with an appropriate volume for subsequent staining with fluorescently labelled antibodies. Lymphocytes were mechanically isolated from draining lymph nodes with a pestle, filtered through a 70-µm filter and resuspended as a single-cell suspension with an appropriate volume for subsequent staining with fluorescently labelled antibodies.