Non-parenchymal cells (NPCs) were isolated from liver lobes by perfusion with Collagenase H (Sigma, St. Louis, MO, USA) followed by density centrifugation as previously described (25 (link), 26 (link)). Non-parenchymal cells (NPCs) were immuno-labeled with fluorochrome-conjugated antibodies (eBiosciences and BioLegend) followed by flow cytometry analysis (LSR Fortessa X20™, BD) or cell sorting (FACSAria, BD). Antibodies are listed in Supplementary Methods .
Collagenase h
Manufactured by Merck Group
Sourced in United States, United Kingdom
Collagenase H is an enzyme used in laboratory settings to isolate and extract cells from tissue samples. It functions by breaking down the collagen-based extracellular matrix, facilitating the release of cells for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Dnase, by Roche (4 mentions)
Cesium chloride, by Merck Group (4 mentions)
Nifedipine, by Merck Group (4 mentions)
Mgatp, by Merck Group (4 mentions)
Papain, by Worthington (4 mentions)
Elastase, by Worthington (4 mentions)
Papain, by Merck Group (3 mentions)
Ca2 mg2 free phosphate buffered saline, by Merck Group (3 mentions)
40 μm cell strainer, by Merck Group (3 mentions)
Niflumic acid, by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Tetraethylammonium chloride, by Merck Group (2 mentions)
Trypsin inhibitor from glycine max, by Merck Group (2 mentions)
Collagenase f, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Wisent (2 mentions)
Trypsin inhibitor, by Merck Group (2 mentions)
Penicillin streptomycin, by Wisent (2 mentions)
28 protocols using collagenase h
1
Isolation and Flow Cytometry of Liver NPCs
2
Cell Viability and Cytotoxicity Assay
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After the 22-h seeding periods, supernatants were recovered and centrifuged at 300×g for 10 min for each seeding techniques. Cell-seeded PETG rods were incubated with trypsin 0.05% (Fisher Scientific)/EDTA 0.01% (Teknisciences) for 10 min to let the cells detach, and then centrifuged at 300×g for 10 min. The recovered cells from supernatant and scaffolds were incubated for 15 min with Calcein AM 2.5 nM (Thermo-Fisher) to label live cells and Ethidium homodimer-1 4 μM (Thermo-Fisher) to label dead. Cells were immediately analysed with a BD FACSMelody™ flow cytometer (BD Biosciences) and data were treated using the FlowJo™ v9 software (Ashland, OR, USA). For mature tissues, they were first recovered from the scaffolds and placed in a digestion solution of 5.7U/ml collagenase H (Sigma-Aldrich) in accutase (Sigma-Aldrich) at 37 °C with 300 rpm agitation for 30 min. Cells were filtered through a 40 μm cell strainer (Fisher Scientific) and centrifuged at 300×g for 10 min. Finally, cells were analysed as described below.
3
Characterization of Antitumor Immune Responses in Mice
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On day 15 or 16 post-tumor challenge, mice were euthanized and tumors, spleens, and tumor-draining lymph nodes (LNs) were collected to characterize the cell-mediated antitumor immune responses. Briefly, tumors were digested in complete RPMI media containing Collagenase H (Sigma-Aldrich, St. Louis, MO, USA) and DNase (Roche, Indianapolis, IN, USA), and they were incubated at 37 °C for 45 min before passing through a 70 μm cell strainer. Tumor-infiltrating leukocytes (TILs) were isolated using 67%:44% Percoll (Cytiva, Marlborough, MA, USA) gradient centrifugation. Single-cell suspensions from the spleens were prepared using mechanical disruption and passing through cell strainers followed by red blood cell lysis. In mice with mEER oral tumors, the cervical lymph nodes were collected, and in mice with TC-1 vaginal tumors, the inguinal lymph nodes were collected. In both cases, these tumor-draining lymph nodes were processed using mechanical disruption through a 70 μm cell strainer followed by centrifugation and re-suspension in complete RPMI medium.
4
Ion Channel Modulation Assay
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Nifedipine, NA, tetraethylammonium chloride (TEA), ACH, pyrazole-3 (Pyr3), bovine serum albumin (BSA), papain, collagenase H, dithiothreitol (DTT), dithioerythritol (DTE), cesium acetate, cesium chloride (CsCl), Mg-ATP, and agarose were purchased from Sigma (St. Louis, MO, USA). Nifedipine and Pyr3 were dissolved in dimethyl sulfoxide (DMSO), and other compounds were dissolved in the appropriate solutions used in the experiments.
5
Isolation and Culture of Endothelial Cells
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Cells were isolated from adult carotid arterial segments migrated from the cut end of the explants in 2–4 days of culture, and these were collected from the collagen gel by digestion with collagenase H (Sigma-Aldrich Inc.). The endothelial cells were purified by pulldown using CD31 and CD34 antibodies following the published protocol [9 (link)]. The collected cells were cultured in DMEM on a collagen matrix supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in 5% CO2 and room air.
6
Diacerein Modulation of Ionic Channels
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Diacerein was purchased from GuoDa Drugstore (Shanghai, China) and dissolved in deionized distilled water for further use. Acetylcholine chloride (ACh) was purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China). Bovine serum albumin (BSA), cesium chloride (CsCl), collagenase H, dithiothreitol (DTT), gadolinium, MgATP, nifedipine, niflumic acid (NA), ovalbumin (OVA), papain, paxilline (PAX), pyrazole 3 (Pyr3), and tetraethylammonium chloride (TEA) were purchased from Sigma (St. Louis, MO, USA). TRIzol® was purchased from Invitrogen (Carlsbad, CA, USA). RNA extraction kit and complementary DNA (cDNA) synthesis kit were purchased from Takara (Otsu, Japan). SYBR® Green Realtime PCR Master mix was purchased from Toyobo (Osaka, Japan). All other chemicals were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China).
7
Isolation of Mesenteric Artery Myocytes
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Third- and fourth-order mesenteric arteries were carefully cleaned of surrounding adipose and connective tissues, dissected, and held in ice-cold dissecting solution (Mg2+-PSS; 5 mM KCl, 140 mM NaCl, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES adjusted to pH 7.4 with NaOH). Arteries were first placed in dissecting solution supplemented with 1.23 mg/ml papain (Worthington Biochemical, Lakewood, NJ) and 1 mg/ml DTT for 14 min at 37°C. This was followed by a second 5 min incubation in dissecting solution supplemented with 1.6 mg/ml collagenase H (Sigma-Aldrich, St. Louis, MO), 0.5 mg/ml elastase (Worthington Biochemical), and 1 mg/ml trypsin inhibitor from Glycine max (Sigma-Aldrich) at 37°C. Arteries were rinsed three times with dissection solution and single cells were obtained by gentle trituration with a wide-bore glass pipette. Myocytes were maintained at 4°C in dissecting solution until used.
8
Pharmacological Modulators of Ion Channels
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Papain, dithioerythritol, dithiothreitol, collagenase H, bovine serum albumin, 2-aminoethoxydiphenyl borate (2-APB), acetylcholine (ACH) and chloroquine (chloro) were purchased from Sigma. Iberiotoxin (IbTx), paxilline (pax), ryanodine and U73122 were purchased from Cayman Chemical. Fluo-4 AM and fura-2 AM were from Molecular Probes.
9
Tumor Isolation and Immune Cell Analysis
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mT4-2D or mT4-LA cells were harvested with 0.05% trypsin, washed with PBS, counted, and resuspended in ice cold PBS containing 30% Matrigel for implantation of 2.5×105 cells in 100 µL subcutaneously or 3.5×104 cells in 50 µL orthotopically as described. Mice were treated with intratumoral CDN), checkpoint blockade antibodies, or chemotherapy according to treatment schedules included in associated figures. Following euthanasia, primary tumors were harvested, massed, and finely diced into 70 µm filters within 6 cm petri dishes. Diced tumors were enzymatically digested in a 37oC incubator for 30 min in digestion media consisting of X-Vivo15 media (Lonza) supplemented with collagenase H (1 mg/mL; Sigma) and DNAse (160 µg/mL; Roche), then tumors were physically mashed through 70 µm filters to create single cell suspensions. Total cells were counted, then live immune cells were purified by ficoll gradient centrifugation (Histopaque 1119; Sigma). Samples were fixed overnight and permeabilized using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience/ThermoFisher) for staining with fluorescently labeled antibodies in 96-well U-bottom plates. Stained samples were analyzed using a BD LSRII or X-30 prototype flow cytometer.
10
Biochemical Reagents and Compounds
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HSP (analytical standard), L-glucose, collagenase F, collagenase H, dithiothreitol, acetylcholine chloride (Ach), 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α (U46619), forskolin, 4-aminopyridine (4-AP), Na2ATP, STZ, HEPES, potassium aspartate, sodium carboxmethylcellulose, DMEM, albumin and KCl were all purchased from Sigma-Aldrich; Merck KGaA. Papain was purchased from Worthington Biochemical Corporation. Penicillin G and streptomycin were purchased from Beijing SolarBio Science & Technology Co., Ltd.
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