Gentle collagenase

Manufactured by STEMCELL

Gentle Collagenase is a highly purified enzyme solution designed for the gentle dissociation of cellular aggregates and tissue samples. It effectively breaks down collagen, a key structural component of the extracellular matrix, without compromising cell viability.

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Lab products found in correlation

Dnase 1, by STEMCELL (2 mentions) Anti cd16 32, by BioLegend (2 mentions) Live dead fixable zombie yellow fixable viability kit, by BioLegend (2 mentions) Foxp3 fixation permeabilization buffer set, by BioLegend (2 mentions) Y 27632, by STEMCELL (2 mentions) Anti mouse cd16 cd32 clone 2.4g2, by BD (1 mentions) Anti cd24, by BD (1 mentions) Facscalibur, by BD (1 mentions) Human fcr blocking reagent, by Miltenyi Biotec (1 mentions) Epicult basal medium, by STEMCELL (1 mentions)

4 protocols using gentle collagenase

Mammary Gland Cell Isolation Protocol

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Mice were injected with the indicated virus in the #3 or 4 mammary glands with no greater than 2 replicates of a single condition per mouse. Individual mammary glands were harvested digested according to Stemcell Technologies gentle collagenase/hyaluronidase protocol. Briefly glands we digested overnight shaking at 37^oC in 250 ul gentle collagenase (Stemcell Technologies #07919) in 2.25 ml of complete Basal Epicult media formulated according to manufacture instructions (Epicult Basal Medium Stemcell Technologies #05610, 10% Proliferation Supplement, 5% FBS, 1% Penicillin-Streptomycin, 10 ng/ml EGF, 10 ng/ml bFGF, 0.0004% heparin). Glands were then treated with ammonium chloride and triturated for 2 minutes in pre-warmed trypsin followed by dispase. Cells were stained with CD45, CD31, Ter119, CD49f and EPCAM for luminal and basal cell identification.

Langille E., Al-Zahrani K.N., Ma Z., Liang M., Uuskula-Reimand L., Espin R., Teng K., Malik A., Bergholtz H., El Ghamrasni S., Afiuni-Zadeh S., Tsai R., Alvi S., Elia A., Lü Y., Oh R.H., Kozma K.J., Trcka D., Narimatsu M., Liu J.C., Nguyen T., Barutcu S., Loganathan S.K., Bremner R., Bader G.D., Egan S.E., Cescon D.W., Sørlie T., Wrana J.L., Jackson H.W., Wilson M.D., Witkiewicz A.K., Knudsen E.S., Pujana M.A., Wahl G.M, & Schramek D. (2022). Loss of epigenetic regulation disrupts lineage integrity, induces aberrant alveogenesis and promotes breast cancer. Cancer discovery, 12(12), 2930-2953.

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Phenotypic Characterization of ERBB2/3 in Mammary Cells

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Ba/F3 cells stably expressing empty vector or expression vectors for ERBB2 and ERBB3 were stained with PE‐conjugated antibodies targeting each receptor (BD Biosciences) for 20 minutes in the presence of a human FcR‐blocking reagent (Miltenyi), washed, and then read on a FACS Calibur (BD Biosciences). For mammary gland analysis, pairs of abdominal mammary glands from 6‐week old mice were digested overnight in gentle collagenase (Stem Cell Technologies) per manufacturer's instructions, followed by digestion with trypsin and DNAse I. Cells were stained with an antibody cocktail containing propidium iodide, anti‐mouse CD16/CD32 (clone 2.4G2, BD Biosciences), anti‐CD24 (BD Biosciences), and anti‐CD45, ‐CD31, ‐Ter119, ‐CD29, ‐CD49f, and ‐EpCAM (BioLegend).

Senger K., Yuan W., Sagolla M., Doerr J., Bolon B., Ziai J., Sun K., Warming S., Roose‐Girma M., Zhang N., Tam L., Newman R.J., Chaudhuri S., Antony A., Goldstein L.D., Durinck S., Jaiswal B.S., Lafkas D., Modrusan Z, & Seshagiri S. (2020). Embryonic lethality and defective mammary gland development of activator‐function impaired conditional knock‐in Erbb3 V943R mice. Advanced Genetics, 2(1), e10036.

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Single-Cell Isolation and Sorting of Tumor Cells

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Tumors were minced, chopped, and digested with Gentle Collagenase, 0.012% Dispase (w/v) and DNaseI (STEMCELL Technologies) at 37℃ for 1 hour. Single cell suspensions were obtained by passage through a strainer (70 μm), washed in FACS buffer (PBS with 5% FBS), incubated with LIVE/DEAD Fixable Zombie Yellow Fixable Viability Kit (Biolegend, 423104) for 30 min., and blocked with anti-CD16/32 (Biolegend, clone 93) for 5 min. on ice. Primary fluorophore-conjugated antibodies were added, and samples were incubated on ice for 45 min. FOXP3 Fixation/Permeabilization Buffer Set (BioLegend) was used for intracellular markers, according to the manufacturer’s instructions. Antibodies for flow cytometry are listed in Supplementary Table S2. Flow cytometry was performed on an LSR II flow cytometer at the Flow Cytometry Core of the PCC Precision Immunology Shared Resource and analyzed with FlowJo software. Organoids cultured 6 days after infection with MSCV-Kras-mCherry were collected and digested as above, passed through a strainer (70 μm) to obtain single-cell suspensions, centrifuged at 1000×g for 5 min, and resuspended in PBS containing 2% FBS, 10 μM Y-27632, (STEMCELL Technologies Inc.), and DAPI (1 μg/ml). FACS was performed immediately on a MoFloTM XDP, and mCherry^hi and mCherry^neg cells were seeded at 5,000/well.

Zhang S., Iyer S., Ran H., Dolgalev I., Gu S., Wei W., Foster C.J., Loomis C.A., Olvera N., Dao F., Levine D.A., Weinberg R.A, & Neel B.G. (2020). Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer. Cancer discovery, 11(2), 362-383.

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Isolation and Flow Cytometry of Tumor Cells

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Tumors were minced, chopped, and digested with Gentle Collagenase, Dispase 0.012% (w/v) and DNaseI (STEMCELL Technologies) at 37 for 1 hour. Single cell suspensions were obtained by passage through a strainer (70 µm), washed in FACS buffer (PBS with 5% FBS), incubated with LIVE/DEAD Fixable Zombie Yellow Fixable Viability Kit (Biolegend, 423104) for 30 min., and then blocked with anti-CD16/32 (Biolegend, clone 93) for 5 min. on ice. Samples were then incubated with primary fluorophore-conjugated antibodies on ice for 45 min. For detection of intracellular markers, FOXP3 Fixation/Permeabilization Buffer Set (BioLegend) was used, according to the manufacturer's instructions. Antibodies for flow cytometry are described in the Star Methods. Flow cytometry was performed on an LSR II flow cytometer at the Flow Cytometry Core of the PCC Precision Immunology Shared Resource and analyzed with FlowJo software. Organoids cultured 6 days after infection with MSCV-Kras-mCherry were collected and digested as above, passed through a strainer (70 µm) to obtain single-cell suspensions, centrifuged at 1000×g for 5 min, and resuspended in PBS containing 2% FBS, 10 µM Y-27632, (STEMCELL Technologies Inc.), and DAPI (1 µg/ml). FACS was performed immediately on a MoFloTM XDP, and mCherry hi and mCherry neg cells were seeded at 5,000/well.

Zhang S., Iyer S., Ran H., Dolgalev I., Wei W., Foster C., Weinberg R.A., & Neel B.G. (2020). Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer.

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