Dispase 2

Manufactured by Thermo Fisher Scientific

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Dispase II is a proteolytic enzyme derived from Bacillus polymyxa. It is commonly used for the dissociation of a variety of cell and tissue types, including epithelial, endothelial, and fibroblast cells, to facilitate cell isolation and tissue disaggregation.

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257 protocols using dispase 2

Isolation and Culture of RPE Cells

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RPE cells were isolated according to the method reported by Shang et al. [24 (link)], with some modifications. The enucleated eyes were digested for 35 min at 37 °C in dispase II solution (2 % dispase II [Thermo Fisher Scientific], 10 mM HEPES [pH 7.4], 30 mM NaCl in DMEM [4.5 g/L glucose]). Digested eyeballs were washed twice with growth medium (DMEM [4.5 g/L glucose] containing 10 % FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2.5 mM l-glutamine). An incision was made near the ora serrata of the eyes and the anterior segment of the eye was removed. The remaining posterior eye was transferred to a new growth medium, and the neurosensory retina (NSR) was removed. The RPE–choroid complex was transferred to a new growth medium and RPE cell sheets were peeled from the choroid. Isolated RPE cells were washed twice with growth medium and centrifuged (800×g, 4 °C, 5 min).

Mori R., Abe M., Saimoto Y., Shinto S., Jodai S., Tomomatsu M., Tazoe K., Ishida M., Enoki M., Kato N., Yamashita T., Itabashi Y., Nakanishi I., Ohkubo K., Kaidzu S., Tanito M., Matsuoka Y., Morimoto K, & Yamada K.I. (2024). Construction of a screening system for lipid-derived radical inhibitors and validation of hit compounds to target retinal and cerebrovascular diseases. Redox Biology, 73, 103186.

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Isolation and Labeling of Kidney Cells

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We used a previously published protocol (10 (link)) where kidney tissues were fixed with 4% paraformaldehyde and protease inhibitors for 30 minutes prior to permeabilization; they were then incubated with collagenase type I (Thermo Fisher Scientific) and dispase II (Invitrogen) for 1 hour. Samples were passed through a 16.5 gauge needle; then, they were passed through a 20 gauge needle and a 50 μM filter. After centrifugation at 5000g for 5 minutes at 4°C, samples were incubated with Fc blocking antibody (CD16/CD32 rat anti-mouse, clone 2.4G2, BD Biosciences) followed by anti-GFP (Novus Biologicals, catalog NB600-308), anti–rabbit Alexa Fluor 488 (Cell Signaling Technology, catalog 4412S), and DAPI (Cell Signaling Technology). Data were acquired with BD LSR Fortessa Analyzer at the VUMC Flow Cytometry Shared Resource and analyzed by FlowJo (Becton Dickinson).

Osaki Y., Manolopoulou M., Ivanova A.V., Vartanian N., Mignemi M.P., Kern J., Chen J., Yang H., Fogo A.B., Zhang M., Robinson-Cohen C, & Gewin L.S. (2022). Blocking cell cycle progression through CDK4/6 protects against chronic kidney disease. JCI Insight, 7(12), e158754.

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Evaluating Organoid Response to Targeted Therapies

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Five days after organoid trypsinization, 1 mg/ml dispase II (Invitrogen) was added to the medium of the organoids and these were incubated for 15 min at 37° C to digest the BME. Subsequently, organoids were mechanically dissociated by pipetting, filtrated using a 40 μm nylon cell strainer (Falcon), resuspended in 75% BME/growth medium (40 organoids/μl) prior plating of two 10 μl drops on Nunc™ Lab-Tek™ II Chamber Slide™ Systems. After plating culture medium containing either 1 μM of afatinib, 1 μM of selumetinib, a combination of 1 μM of afatinib and 1 μM of selumetinib or DMSO was added. The labtek plates were mounted on an inverted confocal laser scanning microscope (Leica SP8X) and imaged using a 10X objective. For visualization of cell viability, organoids were incubated with 16.2 μΜ Hoechst 33342 (Life Technologies) and 1.5 μM DRAQ7™ (Cell Signaling #7406) for 30 min at 37° C prior imaging.
For the GAP domain knock out CRISPR screen, organoids were imaged by an inverted routine microscope (Nikon Eclipse TS100) using a 4X or 10X objective. For calculating organoid count and size, organoids were incubated for 45 minutes with 500 ml culture medium containing 5 μM calcein-green (Invitrogen). For the quantification of the organoid size and count, FIJI analysis software was used.

Post J.B., Hami N., Mertens A.E., Elfrink S., Bos J.L, & Snippert H.J. (2019). CRISPR-induced RASGAP deficiencies in colorectal cancer organoids reveal that only loss of NF1 promotes resistance to EGFR inhibition. Oncotarget, 10(14), 1440-1457.

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Isolation and Purification of Pterygium Cells

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Freshly collected recurrent pterygium tissues were cut into small sections, washed in Hanks solution, and incubated in DMEM medium (Invitrogen, Carlsbad, California, US) with 100 mmol/L sorbitol (Sigma, Missouri, US) and 50 μg/mL dispase II (Invitrogen) for 60 minutes at 37°C. Then, pterygium cells were dissociated using TryPLE reagent (Invitrogen) for 5 minutes at 37°C. The isolated cells were cultured in DMEM medium supplemented with 10% FBS and gentamicin (50 g/mL) at 37°C in a humidified atmosphere with 5% CO₂.
Purification of cell: First, we used flow cytometry to separate p63 (+) and pan cytokeratin (+) pterygium cells.¹², ¹³, ¹⁴ Next, we observe the cell morphology under an inverted phase‐contrast microscope (Olympus, Tokyo, Japan) and scrape off other cells using cell scraper. Stable cells passaged for 3 to 7 generations were used in the experiments. C646 (Selleck Chemicals, Houston, TX, USA) was used at 10 μmol/L for 48 hours as required.

Gao Y., Luo X, & Zhang J. (2020). Activation of LncRNA FOXD2‐AS1 by H3K27 acetylation regulates VEGF‐A expression by sponging miR‐205‐5p in recurrent pterygium. Journal of Cellular and Molecular Medicine, 24(24), 14139-14151.

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Quantification of Platinum-DNA Adducts

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Platinium-DNA adduct measurement was performed using inductively coupled plasma-mass spectrometry exactly as previously described [21 (link)]. Briefly, organoids were treated with DMSO or with oxaliplatin for 5 h or 24 h in the presence or absence of 2 mM of reduced GSH (Sigma). Organoids were released from the Matrigel^®/BME matrix by addition of 1 mg/ml dispase II (Invitrogen) for 15–30 min at 37 °C. Organoid pellets were lysed for genomic DNA extraction using DNeasy Blood & Tissue kit from Quiagen according to the manufacturer’s instructions.

Laoukili J., Constantinides A., Wassenaar E.C., Elias S.G., Raats D.A., van Schelven S.J., van Wettum J., Volckmann R., Koster J., Huitema A.D., Nienhuijs S.W., de Hingh I.H., Wiezer R.J., van Grevenstein H.M., Rinkes I.H., Boerma D, & Kranenburg O. (2022). Peritoneal metastases from colorectal cancer belong to Consensus Molecular Subtype 4 and are sensitised to oxaliplatin by inhibiting reducing capacity. British Journal of Cancer, 126(12), 1824-1833.

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Culturing and Differentiating Murine Myoblasts

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The C3H murine skeletal muscle cell line C2C12 myoblasts (American Type Culture Collection, CRL‐1772) were cultured in growth medium (GM), which was consisting of Dulbecco's modified Eagle's medium (DMEM) (cat. no. 10569‐010, Gibco), 10% FBS (cat. no. 10099–141, Gibco) and 1% penicillin‐streptomycin (cat. no. SV30010, Hyclone) at 37°C under a humidified atmosphere with 5% CO₂. Primary myoblasts were isolated from the limbs of 2‐ to 5‐day‐old C57BL/6 mice. The limb tissue was firstly digested with 1% type II collagenase (cat. no. 17101‐015, Invitrogen), 2.4 U/ml dispase II (cat. no. 42613‐33‐2, Sigma‐Aldrich) and 2.5 mM CaCl₂ in PBS for 30 min at 37°C. The digested tissue block was screened by nylon mesh filter (100 μm). After centrifugation, the isolated myoblasts were cultured in DMEM supplemented with 30% FBS and 1% penicillin‐streptomycin at 37°C with 5% CO₂ atmosphere. For differentiation of muscle cells into myotube, the GM was replaced with differentiation medium (DM) containing DMEM supplemented with 2% horse serum (cat. no. 26050088, Gibco) and 1% penicillin‐streptomycin when C2C12 cells and primary myoblasts at 90% confluence. The DM was changed every 24 h and detected in the indicated number of differentiation days. All experimental groups of cell culture were performed at least in triplicate.

Song R., Zhao S., Xu Y., Hu J., Ke S., Li F., Tian G., Zheng X., Li J., Gu L, & Xu Y. (2021). MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration. Journal of Cellular and Molecular Medicine, 25(18), 8645-8661.

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Isolation of Primary Mouse Meibocytes

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The preparation of primary mouse meibocytes was described in a previous study [33 (link)]. Briefly, the upper and lower eyelids of twenty 4-week-old mice were removed, disinfected with 75% ethanol, and dissected under a Zeiss microscope. The tissues were washed with PBS and placed in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/mL penicillin/streptomycin (Gibco, USA). After the conjunctiva and excess skin were removed, the tarsal plates were placed in DMEM containing 0.25% collagenase A (Invitrogen, Carlsbad, CA) and 0.6 U/mL dispase II (Invitrogen). Then, the glands were digested at 37 °C for 6 h, followed by centrifugation at 1000 rpm for 10 min. Subsequently, the cell pellet was suspended in keratinocyte growth medium (KGM, Lonza Walkersville, Inc., Walkersville, MD, USA) containing human epidermal growth factor (hEGF), hydrocortisone, insulin and gentamicin and supplemented with 10% fetal bovine serum (FBS). Cells were cultured in 6-well plate in a humidified atmosphere with 5% CO₂ at 37 °C.

Chen L., Yan D., Wu N., Yao Q., Sun H., Pang Y, & Fu Y. (2021). Injectable bio-responsive hydrogel for therapy of inflammation related eyelid diseases. Bioactive Materials, 6(10), 3062-3073.

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Isolation of Primary Murine Keratinocytes

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Tails of littermates were wiped with 70% Ethanol, and then removed at base and carefully incised using a scalpel to make a longitudinal incision down the tail; skin was subsequently removed from the tails using tweezers. Skin was then incubated overnight at 4°C in keratinocyte serum free media (from Invitrogen) with dispase II at a final concentration of 2.1 U/ml to cleave epidermal–dermal junctions. The next day, epidermis was separated from dermis. Epidermis was then incubated for 15 min in 2 ml TrypLE express trypsin (from Invitrogen) to isolate keratinocytes. After addition of medium, cells were centrifuged at 600 rpm for 10 min. Supernatant was removed and the pellet was resuspended in medium supplemented with bovine pituitary extract and epidermal growth factor, including gentamicin at 50 µg/ml (Sigma-Aldrich). Floating dead cells were removed the next day. Keratinocytes were then grown to 70–90% confluency until further experiments were performed, usually after 3–5 additional days in culture.

Zinngrebe J., Rieser E., Taraborrelli L., Peltzer N., Hartwig T., Ren H., Kovács I., Endres C., Draber P., Darding M., von Karstedt S., Lemke J., Dome B., Bergmann M., Ferguson B.J, & Walczak H. (2016). LUBAC deficiency perturbs TLR3 signaling to cause immunodeficiency and autoinflammation. The Journal of Experimental Medicine, 213(12), 2671-2689.

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Isolation and Culture of Thyroid Cancer Cells

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Cancer tissues obtained from mPTC^GFP were minced in lysis buffer (Collagenase I (Sigma) 1 mg/ml; Dispase II (Invitrogen) 0.5 mg/ml in PBS) and shaked on an orbital shaker at 37 °C for 60 min. Dissociated tumor cells were filtered with 40 μm cell strainer (#352340, BD Falcon) and centrifugated at 500 g for 5 min, the pellet was resuspended in cold Ham’s F-12 Nutrient Mix (#11765062, Gibco). The isolation of tumor cells was next performed on FACSAria Cell Sorter (Becton Dickinson) according to GFP signal, and positive cells were washed with PBS and then cultured in F-12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 5 mg/L transferrin (Sigma-Aldrich), 10 mg/L bovine insulin (Solarbio), 3.5 mg/L hydrocortisone (Sigma-Aldrich), 10 mg/L somatostatin (Sigma-Aldrich), 0.02 mg/L Gly-His-Lysacetate (Sigma-Aldrich) and 1 IU/L bovine thyroid stimulating hormone (TSH) (Sigma-Aldrich) related to the 6H medium at 37 °C⁵⁹. Next, Primary cells treated with 10 μM PLX4032 or 2 μM SCH772984 were subjected to western blot and RT-qPCR analysis. Primary cells from mPTC mice were directly cultured in complete F-12 medium. Thyrocytes were confirmed by immunofluorescence staining of thyroglobulin and representative photos were taken by a fluorescence microscopy.

Zhang P., Guan H., Yuan S., Cheng H., Zheng J., Zhang Z., Liu Y., Yu Y., Meng Z., Zheng X, & Zhao L. (2022). Targeting myeloid derived suppressor cells reverts immune suppression and sensitizes BRAF-mutant papillary thyroid cancer to MAPK inhibitors. Nature Communications, 13, 1588.

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Isolation of Limbal Epithelial Sheets

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Human corneoscleral rims from donors younger than 50 years were obtained from discarded tissue after keratoplasties performed at the MEEI. The removal of limbal epithelial sheets was performed as previously described [14 ]. In short, after corneoscleral tissue was rinsed with Hanks’ balanced salt solution, the sclera, conjunctiva, iris, trabecular meshwork, and corneal endothelium were removed. Then the limbal tissue was cut into 3-clock-hour segments and placed in 1.2 IU of Dispase II (Invitrogen) for 1 hour at 37°C. Intact epithelial sheets were removed from stroma and de-epithelialized stroma was further digested with 0.025% Trypsin/EDTA (Invitrogen) for 30 minutes at 37°C to yield single cells. Stromal cell suspension was then filtered with 70 μm nylon filter, and cells were resuspended in KnockOut ESC/iPSC medium on thin-coated Matrigel 6 well plates (BD Labware; Bedford, MA, http://www.bd.com) for 12–15 days (Fig. 1).

Katikireddy K.R., Dana R, & Jurkunas U.V. (2014). Differentiation Potential of Limbal Fibroblasts and Bone Marrow Mesenchymal Stem Cells to Corneal Epithelial Cells. Stem cells (Dayton, Ohio), 32(3), 717-729.

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