All experiments using Xenopus and zebrafish were done with approval of the HMS animal care review board. Ovaries were surgically removed from adult female Xenopus laevis frogs and treated with 2 mg/mL collagenase 1A (Sigma) in 1X MMR by gentle rocking, until most of the oocytes were clearly dissociated. Oocytes were later injected with mRNAs encoding for indicated proteins by using a FemtoJet express microinjector (Eppendorf).
Collagenase 1a
Manufactured by Merck Group
Sourced in United States, Germany
Collagenase 1A is an enzyme that breaks down collagen, a structural protein found in various tissues. It is commonly used in cell culture and tissue engineering applications to facilitate the isolation and dissociation of cells from their extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Trypsin, by Lonza (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Corning (2 mentions)
Fluorescently tagged antibodies, by BioLegend (2 mentions)
Penicillin streptomycin, by BD (2 mentions)
Countbright beads, by Thermo Fisher Scientific (2 mentions)
Fortessa, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Dnase 1, by Worthington (2 mentions)
Dnase 1, by Roche (2 mentions)
38 protocols using collagenase 1a
1
Xenopus Oocyte Preparation and mRNA Injection
2
Gingival Biopsies and Saliva Collection from Healthy and HIV+ Cohorts
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Human samples (gingival biopsies and saliva) were obtained with informed consents from healthy and HIV+ (PLWH) cohorts under a protocol approved by the University Hospitals Cleveland Medical Center Institutional Review Board, complying with all relevant ethical regulations4 (link). Participants of both sexes were enrolled. The characteristics of enrolled participants for obtaining gingival biopsies and saliva are described in Supplementary Table 1 . Gingival biopsies were processed fresh for flow cytometry. Discarded palatine tonsils were obtained from tonsillectomy surgeries performed at University Hospitals Cleveland Medical Center through the Histology Tissue Procurement Facility following a separate IRB-approved protocol (Non-Human research). A single-cell suspension of gingival tissues and tonsils was prepared by Collagenase 1A digestion (0.5 mg/ml; Sigma C9891), with subsequent Ficoll-Paque PLUS (GE17-1440-02; Millipore Sigma) centrifugation at 900 g and washing with PBS. Tonsil cells were processed fresh for HTOC cultures. Some tonsil cells were processed fresh for CD4 cell purification for cell culture or flow cytometry. Saliva samples were collected in sterile tubes and stored at −80 °C until processing for metabolome and ELISA analyses.
3
Single-Cell Isolation from Tumor Tissue
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Following resection, a random piece was selected from viable tumor tissue, minced, and viably frozen. On the day of the sorting, the sample was thawed and dissociated into a single-cell suspension in AdDF + ++ (Advanced DMEM/F12 containing 1× Glutamax, 10 mM HEPES and antibiotics) containing Collagenase 1a (1 mg/mL, Sigma, C9407) and DNase (0.25 µg/mL, Stemcell), supplemented with Rho-kinase inhibitor Y-27632 (10 µM, Abmole). The samples were digested on an orbital shaker for 30 min at 37 °C. The suspension was washed first with AdDF + ++ and next with MACS buffer (PBS pH 7.2 + 2 mM EDTA + 0.5% Bovine Serum Albumine), followed each time by centrifugation at 300 × g. Viable single cells were sorted based on forward/side scatter properties and DAPI/DRAQ5 staining using FACS (MoFlo Astrios EQ, Beckman Coulter) into 384-well plates (Biorad) containing 10 µl mineral oil (Sigma) and 50 nl of RT primers.
4
PDX Tumor Dissociation and Co-culture Assay
5
Pharmacological Assays with Key Reagents
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Nystatin, angiotensin II, vasopressin, endothelin, heptanol 18BGRA, collagenase 1A, protease XIV and other chemicals were from Sigma (St Louis, MO). Reagents were thawed and diluted on the day of the experiment and excess discarded daily.
6
Chondrocyte Isolation and Characterization
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Articular cartilage was removed from the bone using a scalpel and digested with collagenase 1 A (Sigma Aldrich) (1 mg/ml) for 6 h. Digested tissue was passed through a 70 µm cell strainer to remove debris and the flow-through was centrifuged at 2,000 × g for 5 min. The resulting cell pellet was resuspended in chondrocyte culture medium (DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 1% v/v non-essential amino acids (Sigma Aldrich)). The phenotype of the chondrocytes was confirmed by positive Type II collagen expression by Western Blotting (Supplementary Fig. 2 ).
7
Senescence Detection in Tumor Samples
8
Isolation and Staining of Pancreatic Cells
9
3D Tumor Spheroid Culture Optimization
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For one spheroid, 30,000 cells were cultured in 100 μL of medium, supplemented by 0.25% to 1% of either MethocultTM H4100 or SF H4236 (StemCell, Grenoble, France), and seeded in U-bottomed 96-well plate (Sarstedt, Marnay, France). Both media contains methylcellulose in IMDM, but SF H4236 is supplemented with bovine serum albumin, recombinant human insulin, human transferrin (iron-saturated), 2-Mercaptoethanol and unknown supplements as described by the manufacturer. The medium was the same as that of the normal culture for each cell line but supplemented with heat inactivated FBS to reach 15%. At days as detailed, microscopic analysis was performed using a Leica DMIL microscope (Leica, Nanterre, France), coupled to a DXM1200F camera (Nikon, Champigny-sur-Marne, France). To determine the number of cells in each spheroid over time, only wells with a unique, fully-formed spheroid were selected. Twelve spheroids per experiment were pooled and dissociated with 2 mg/mL collagenase 1A (Sigma-Aldrich, Saint-Quentin-Fallavier, France), 10 min at 37°C, with agitation every two minutes, and then counted by the trypan blue exclusion assay.
10
Chicken Embryo Tumor Xenograft Assay
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