Soybean trypsin inhibitor

Mammalian reovirus (type 3 Dearing) and RSV (serotype A, Long strain) were propagated, purified, and UV‐inactivated as previously described 28, 31, 32. UV inactivation was confirmed by standard plaque assay and flow cytometric analysis of virus treated cells. Reovirus adsorption was performed by incubating CBMCs at 5 × 10⁶ cells/ml with reovirus at 20 multiplicities of infection (MOI) in activation medium (RPMI 1640 containing 1% FBS, 15 mM HEPES and 10 ng/ml hSCF) for 1 h at 37°C. Medium alone (Mock) or UV‐Reo at MOI 20 were included as controls. The cells were washed and resuspended at 1 × 10⁶ cells/ml in fresh activation medium plus 1% FBS and 100 μg/ml soybean trypsin inhibitor (Sigma). RSV adsorption was performed by incubating CBMC (10⁶/ml) for 90 min at 4°C with RSV at MOI 3–4 in activation media containing 2.5% FBS and 100 μg/ml soybean trypsin inhibitor (Sigma). Medium alone (Mock) or UV‐RSV at MOI 3–4 were included as controls. In some experiments, CBMC were resuspended in activation media containing 5 μg/ml anti‐human anti‐IFN‐α/β receptor chain 2 antibody (clone MMHAR‐2, EMD Millipore) or mouse IgG2a isotype control (clone, MG2a‐53, BioLegend). Cell‐free supernatants were harvested by centrifugation. Cell pellets were lysed and stored for mRNA extraction.

Tissues collected for flow cytometry were maintained in dishes containing 10% FBS MEM on ice for the duration of the mouse harvest to maintain cell viability. Following harvest, tissues were rinsed in HBSS to remove any serum from the samples, since serum can interfere with the tissue digest. Digest solution was made from 5 mg/mL Elastase (Worthington), 0.2 mg/mL Soybean Trypsin inhibitor (MilliporeSigma), and 3.2 mg/mL collagenase II dissolved in HBSS and filtered through a 0.2 mm syringe filter. Individual tissues were minced using dissection scissors in Eppendorf tubes with 500 μL digestion buffer before being incubated in a 37°C incubator for about 1 hour to create a single-cell suspension. Samples were removed from the incubator at approximately 10-minute intervals and pipetted with a p1000 tip to facilitate tissue digestion.
At the end of the digest period, samples were spun down for 12 minutes at 172g (4°C) to pellet the single cells. Supernatant was removed, and cells were resuspended in FA3 Buffer. The FA3 buffer was prepared with 1× PBS, 1 mM EDTA, 25 mM HEPES (pH 7.0), and 1% FBS and was then sterile filtered. Samples were centrifuged again with the same parameters, resuspended FA3, then filtered through 70 mm FLOWMI cell strainers.

The cGMP hydrolysis was measured in cell extracts obtained from HEK293T cells 48 hours after transfection. Where indicated, samples were treated with 0.1 mg/ml TPCK-Trypsin (MilliporeSigma) on ice for 10 minutes to selectively degrade Pγ, after which trypsin was inhibited with the addition of 10-fold excess of soybean trypsin inhibitor (MilliporeSigma) and incubated for 5 minutes at 25°C. Cell extracts (protein concentration 3–6 mg/ml) were diluted 4–400 fold into 40 μl (final volume) of 20 mM Tris–HCl (pH 7.5) buffer containing 120 mM NaCl, 2 mM MgSO₄, 1 mM 2-mercaptoethanol, 0.1 U bacterial alkaline phosphatase, and 10 μM [³H]cGMP (100,000 cpm) (PerkinElmer) for 15 minutes at 37°C. The reaction was stopped by the addition of AG1-X2 cation exchange resin (0.5 ml of 20% bed volume suspension). Samples were incubated for 6 minutes at 25°C with occasional mixing, and spun at 10,000g for 3 minutes. A quantity of 0.25 ml supernatant was removed for counting in a scintillation counter.

Freshly excised tumor slices were obtained from KPCT and KPCG mice. Live cell imaging of tumor slices was performed similar to time-lapse MPE methods described previously (27 (link)). Briefly, the tumor was sliced into a few thin sections (300–350 µm) using a vibratome, set on a 35 mm dish with a slice anchor (Warner Instruments), and overlaid with L-15 media supplemented with 10% FBS, penicillin-streptomycin, plasmocin, fungizone, and 10 μg/mL soybean trypsin inhibitor (MilliporeSigma). Imaging was subsequently performed on the multiphoton microscopy setup described above with a custom-built temperature-controlled stage insert (60 (link)) for 6–12 hours with a time interval of 20 minutes between frames at 880–900 nm excitation wavelength and emission captured in the green (KPCG), red (KPCT), or blue (SHG) channels. Analysis and 3D rendering of Z stacks, visualization of time-lapse imaging data, and image processing were done in Fiji. For live imaging, Z stacks were generally obtained at a step size of 4–5 μm, covering a total depth of about 80–100 μm.

For primary IECs isolation, intestines were opened longitudinally and washed in Ca²⁺ and Mg²⁺-free Hanks’ balanced salt solution (HBSS) containing 2% glucose, 25 ng of amphotericin B/ml, 100 U of penicillin/ml, and 100 mg of streptomycin/ml, which were cut into 1-mm fragments and incubated for 10 min at 22 °C on a shaker platform in HBSS containing collagenase XIa (Sigma), 2% bovine serum albumin, and 0.2 mg of soybean trypsin inhibitor/ml (Sigma). Cells and small sheets of intestinal epithelium were separated from the denser intestinal fragments by harvesting supernatants after two 60-s sedimentations in medium containing Dulbecco’s modified Eagle medium (DMEM) (Gibco), 10% sorbitol (Gibco), 100 U of penicillin/ml, 100 mg of streptomycin/ml, and 5% fetal bovine serum. Cells were centrifuged five times at 1200 g for 3 min in DMEM plus 2% sorbitol. Supernatants were discarded, and the cells were cultured in 10-cm plates coated with 40 ml of Matrigel per cm² diluted 1:2 in phenol-red-free DMEM (Sigma).

Trypsin (Sigma) at a concentration of 1 mg/ml in PBS was added to the permeabilized cells to yield a dilution of 1 × 10⁶ IE equivalents/μl. As a control, mock treated cells without Trypsin were used and treated with PBS only. The samples were incubated for 30 min at 37°C and the reaction was stopped by the addition of 1 mg/ml soybean-Trypsin inhibitor (Sigma) and incubation for 5 min on ice. After centrifugation at the appropriate conditions indicated above for each of the permeabilization methods, the supernatants were discarded and the pellet fraction extracted in 2× protein loading buffer and analysed by western blot analysis.