Primary CNS neurons used in the manuscript were prepared from forebrains of embryonic day 16 (E16) Sprague-Dawley rats [31 (link)]. Forebrains free of meninges were dissected in ice-cold dissection buffer (Ca2+/Mg2+-free Hank’s Balanced Salt Solution containing 10 mM HEPES) (Invitrogen, Carlsbad, CA, USA), dissociated with L-cysteine activated papain (10 units/mL) (Sigma Aldrich, St. Louis, MO, USA) for 5 min at 37 °C, and resuspended in dissection medium containing trypsin inhibitor (10 mg/mL) (Invitrogen, Carlsbad, CA, USA) for 2–3 min followed by washing with the trypsin inhibitor solution. The tissue was resuspended in a plating medium (NBB27 + glutamate: neurobasal medium containing 2% B27, 1 mM Glutamine, 25 μM glutamic acid, 100 units/mL penicillin, and 100 μg/mL streptomycin) (Invitrogen, Carlsbad, CA, USA) and triturated with a fire-polished glass Pasteur pipette. The cells were then passed through a 70 μm cell sieves and live cells were counted using a hemocytometer and trypan blue exclusion assay.
Trypsin inhibitor
Manufactured by Thermo Fisher Scientific
Sourced in United States, Palestine, State of
Trypsin inhibitor is a laboratory reagent used to inactivate the enzymatic activity of trypsin, a proteolytic enzyme commonly used in cell culture and other biological applications. It functions by binding to and inhibiting the catalytic site of trypsin, preventing it from cleaving peptide bonds in proteins.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Trypsin edta, by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Trypsin, by Thermo Fisher Scientific (6 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (6 mentions)
Glutamax, by Thermo Fisher Scientific (6 mentions)
Collagenase d, by Roche (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Insulin, by Merck Group (5 mentions)
Collagenase type 1, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Tryple express, by Thermo Fisher Scientific (4 mentions)
Akc lysis buffer, by Corning (4 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (3 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Merck Group (3 mentions)
Dnase, by Merck Group (3 mentions)
Laminin, by Merck Group (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
57 protocols using trypsin inhibitor
1
Isolation and Culture of Rat Primary CNS Neurons
2
Isolation and Culture of Primary Rat Neurons
3
Isolation and Culture of Murine Primary Microglia
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BV2 microglia (The Cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco C11995500BT, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco 10270-106) at 37 °C in a 5% CO2/95% air incubator.
Primary microglia were isolated from the cerebral cortices of mice as previously described with minor modifications [24 (link),39 (link)]. Briefly, the cerebral cortices were dissected from ICR mice (Guangxi Medical University, Nanning, China) at postnatal day 2–4. After removing dura mater, the tissues were minced, dissociated with 0.25% trypsin-EDTA (Gibco 25200-072) for 30 min at 37 °C, followed by the addition of 600 U/mL DNase I (Gibco 18047-019) and 0.3 mg/mL trypsin inhibitor (Gibco 17075-011) for 15 min. The pellets were collected after centrifugation and suspended in DMEM/F12 (Gibco 12634010) supplemented with 10% FBS. Fifteen percent bovine serum albumin (BSA) solution was added from the bottom of medium to remove debris by centrifugation. Pellets were collected after centrifugation at 740 g for 4 min and re-suspended in 10% FBS-DMEM/F12. Cells were seeded into 6 cm dishes and cultured at 37 °C with 5% CO2. After 14–17 days, microglia were isolated from mixed glial cultures with mild trypsinization [39 (link)].
Primary microglia were isolated from the cerebral cortices of mice as previously described with minor modifications [24 (link),39 (link)]. Briefly, the cerebral cortices were dissected from ICR mice (Guangxi Medical University, Nanning, China) at postnatal day 2–4. After removing dura mater, the tissues were minced, dissociated with 0.25% trypsin-EDTA (Gibco 25200-072) for 30 min at 37 °C, followed by the addition of 600 U/mL DNase I (Gibco 18047-019) and 0.3 mg/mL trypsin inhibitor (Gibco 17075-011) for 15 min. The pellets were collected after centrifugation and suspended in DMEM/F12 (Gibco 12634010) supplemented with 10% FBS. Fifteen percent bovine serum albumin (BSA) solution was added from the bottom of medium to remove debris by centrifugation. Pellets were collected after centrifugation at 740 g for 4 min and re-suspended in 10% FBS-DMEM/F12. Cells were seeded into 6 cm dishes and cultured at 37 °C with 5% CO2. After 14–17 days, microglia were isolated from mixed glial cultures with mild trypsinization [39 (link)].
4
Site-specific Biotinylation of Influenza Hemagglutinin
5
Collagen I Remodeling Assay
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Collagenase type IA (Sigma, St. Louis, MO, USA), Collagen I from rat tail (Corning, Corning, NY, USA), trypsin inhibitor (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), IMDM (Gibco, Thermo Fisher Scientific), FBS (#F7524; Sigma), preactivated recombinant MMP9 (Sigma), murine EGF (PeproTech, Rocky Hill, NJ, USA), vorapaxar (Adooq Bioscience, Irvine, CA, USA), p1pal‐12 (palmitate‐RCLSSSAVANRS‐NH2; GL Biochem, Shanghai, China), 10058‐F4 (c‐Myc inhibitor; Selleck Chemicals, Houston, TX, USA), ActinGreen 488 ReadyProbes Reagent (Thermo Fisher Scientific), Dolichos Biflorus Agglutinin (DBA)‐rhodamine (RL‐1032; Vector Laboratories, Burlingame, CA, USA).
6
Isolation and Analysis of Liver Macrophages
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Liver macrophages were isolated from mice as described (36 (link)). Briefly, mouse livers were cut into small pieces and digested for 1 hour at 37°C in RPMI 1640 medium containing 0.05% collagenase/dispase (Roche) and 0.01% trypsin inhibitor (Gibco, Thermo Fisher Scientific). The liver suspension was pressed through a 40 μm cell strainer, then centrifuged at 800g for 10 minutes at 4°C. The supernatant was removed and erythrocytes were lysed with red blood cell lysis buffer for 2 minutes at 4°C, then centrifuged at 800g for 10 minutes at 4°C. The cell pellet was resuspended in 40% Percoll gradient (MilliporeSigma, P1644). Cell suspensions were loaded on the top of the 80% Percoll gradient and centrifuged at 376g for 25 minutes at room temperature without brake. The middle layer was suspended with 10 mL of RPMI 1640 medium. Cell suspensions were spun at 376g for 5 minutes at room temperature, and supernatant was removed. Cells were resuspended in 2% FCS and 1 × 106 cells were enumerated. Subsequently, cells were stained with F4/80 (BioLegend, catalog 123107) and CD11b antibody (BioLegend, catalog 101211) for 30 minutes in the dark (0.5 μL/1 × 106 cells). Finally, the cells were washed and suspended in 0.5 mL PBS. The stained cells were analyzed using flow cytometry (BD LSRFortessa). Final calculations were performed by FlowJo software (FlowJo LLC).
7
Cultivation of Human Epithelial Cells
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Normal HEEpiCs were purchased from ScienCell Research Laboratories (CA, USA) via Cosmo Bio Co., Ltd. (Tokyo, Japan). HEEpiCs were grown in epithelial cell medium-2 (EpiCM-2, ScienCell, San Diego, CA, USA) in a humidified atmosphere with 5% CO2 in air at 37°C. Once the cells reached 80% confluence, as observed under an inverted microscope (TCM400FLR, LaboMed America Inc., Fremont, CA, USA), they were passaged with 0.25% (w/v) trypsin and 0.03% (w/v) trypsin inhibitor (Gibco, Darmstadt, Germany). HEEpiCs were seeded at a density of 4.0 × 105 (link) cells per well in 6-well plates (BD Biosciences, Franklin Lakes, NJ, USA) for each assay. Cell density was changed in accordance with well numbers of the microplates used in the experiments.
8
Culturing Monkey Embryonic Stem Cells
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Monkey ESCs were cultured at 37°C and 5% CO2 in the MT-fCFA medium. For passaging, the cells were washed twice with phosphate-buffered saline (PBS) and were treated with 0.01% trypsin/0.004% EDTA (Sigma) for 2 min, followed by pipetting to ensure single-cell dissociation. The single dissociated cells were then mixed with 250 µg/ml of trypsin inhibitor (Gibco) to inactivate trypsin, centrifuged at 180×g for 5 min, and resuspended in the MT-fCFA medium containing 2.4 µM thiazovivin (Wako) and 4.7 µg/ml human fibronectin (BD Biosciences). The cells were plated on collagen type I-coated dishes (IWAKI) at a dilution ratio of 1∶8–1∶10. Cells routinely received fresh medium every day and were passaged when 80–90% confluence was reached, which normally occurred every 2–3 days.
For cryopreservation, single dissociated cells were suspended in STEM-CELLBANKER (ZENOAQ, BLC-3S) [17] (link) and directly frozen at −80°C without a programmed freezer in accordance with the manufacturer's instructions.
For cryopreservation, single dissociated cells were suspended in STEM-CELLBANKER (ZENOAQ, BLC-3S) [17] (link) and directly frozen at −80°C without a programmed freezer in accordance with the manufacturer's instructions.
9
Pharmacological Modulation of Purinergic Signaling
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SCH58261, PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid), MRS1754, CVT6883, catalase-polyethylene glycol (Catalase), Glibenclamide, adenosine deaminase (ADA), U46619 (9,11-dideoxy-11α,9α epoxymethanoprostaglandin F2α), and substance P were all purchased from Sigma–Aldrich (Ann Arbor, MI, USA). Up4A was obtained from Biolog Life Science (Bremen, Germany). SCH58261, MRS1754 and CVT6883 were firstly dissolved in DMSO. All subsequent dilutions (at least 1000 fold) and other drugs were obtained with distilled water. PPADS was protected from light. For the cell culture, Hanks’ balanced salt solution, DMEM medium, fetal bovine serum (FBS), antibiotic-antimycotic, collagenase type I, and trypsin inhibitor were purchased from GIBCO (Carlsbad, CA, USA).
10
Isolation of Pancreatic Tumor Cells
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Pancreatic tumors were minced and dissociated into single cells by sequentially incubating them in 1) dissociation media for 1h at 37C with gentle agitation, 2) Trypsin 0.25% for 10 min at 37°C, and 3) ACK lysis buffer (ThermoFisher) for 2 min at room temperature to remove erythrocytes. Dissociation media was prepared freshly as follows: DMEM supplemented with 1 mg/mL Trypsin inhibitor (Gibco), 1 mg/mL Dispase II (Gibco), 1 mg/mL Collagenase IV (Gibco) and 0.025 mg/mL DNAse (Sigma). Final suspensions were passed through a 100 μm nylon mesh to remove aggregates. For labeling, cells were incubated on ice with Mouse Fc Block (BD Biosciences, 1/200) followed by antigen-specific antibodies, in FACS buffer (PBS with 1mM EDTA and 0.5% BSA). Cells were sorted at the Salk Institute FACS core facility on a BD Influx cell sorter. DAPI (Molecular Probes) was used to exclude dead cells. For labeling, the following antibodies were used: CD45-AF488, EPCAM-AF647, and PDGFRα-PE (Biolegend, 1/200).
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