Collagenase 4 from clostridium histolyticum

The colon was separated from mesenteric fat, and the luminal mucus and fecal matter were removed mechanically at the time of dissection. The colon was transected longitudinally and bisected then washed three times by vortexing followed by transfer into fresh conditioned media (CM; Ca²⁺-free Mg²⁺-free HBSS (Hyclone)/5% FCS (Sigma)). The tissue pieces were incubated with gentle mixing in CM with 1 mM DTT and 5 mM EDTA for 15 min at 37°C and then in CM with 5 mM EDTA for 30 min at 37°C. The tissue was then cut into small pieces and digested in 0.4 mg/mL collagenase IV (from Clostridium histolyticum; Sigma Aldrich), 0.1 mg/mL DNAse (Sigma), and 10% FCS in HBSS (with Ca²⁺ and Mg²⁺, Hyclone) for 30 min at 37°C. After digestion, the samples were homogenized using a syringe and 18 gauge needle and filtered through a 70 μm cell strainer.

Splenocytes of tumor-bearing mice were immediately collected after sacrifice, processed and disaggregated following an enzymatic/mechanic procedure: cut into small pieces, digested with 0.5 mg/ml Collagenase IV from Clostridium histolyticum (Sigma, Cat# C5138) in RPMI without serum for 10 minutes at 37°C in an orbital shaker incubator; cell suspension was passed through a 70-μm cell strainer to remove any undigested tissue. Cells were incubated in ACK lysis buffer (Lonza, Cat#10-548e) for 5 min at room temperature to remove red blood cells; then centrifuged at 4°C, 300 rcf for 5 minutes and the pellet was resuspended in complete RPMI. Splenocytes were cultured in 24 well plates (106/well), treated ex vivo for 24 hours, then incubated for 48 hours with stained CMT167 murine lung cancer cells (2x104) and analyzed by FACS, as described in the previous section.

Atropine, Carbachol, FAEE, Taurolithocholic acid disodium salt, Bovine serum albumin, and Collagenase IV from Clostridium histolyticum were obtained from Sigma-Aldrich (St. Louis, USA); Lipopolysaccharide from Escherichia was purchased from Sigma-Aldrich (Rehovot, Israel). The substrates BOC-Gln-Ala-Arg-7AMC and Z-Arg-Arg-7AMC hydrochloride were obtained from Sigma-Aldrich (Buchs, Switzerland), LDH cytotoxicity assay kit was obtained from G-Biosciences (St. Louis, USA). Hisep LSM 1077, Dulbeccos modified eagle medium, Penstrep, Phosphate buffered saline, Fetal bovine serum were purchased from Himedia laboratories (Mumbai, India). Cytometric bead array (CBA) human inflammatory cytokines kit was purchased from BD Biosciences, USA. Autozyme, an amylase quantification kit was purchased from Accurex Biomedicals (Thane, India); recombinant TNF-α was purchased from Peprotech (Rocky Hill, NJ USA). Polyclonal primary antibodies (anti IL-6, anti-TNF-α, anti-LC3, anti caspase-3) were purchased from Abcam (Cambridge, UK), while anti-amylase antibody was purchased from Santacruz (California, USA). All other chemicals were obtained either from Sigma-Aldrich (Germany, USA) or SDFCL Fine chemicals limited (Mumbai, India).