Dispase

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Dispase is an enzymatic cell dissociation reagent used for the isolation and dissociation of cells from various tissue types, including epithelial, endothelial, and connective tissues. It functions by breaking down extracellular matrix proteins, allowing for the efficient release of cells from their surrounding matrix.

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Dispase enzyme Dispase II solution Dispase/DNAse Collagenase IV–dispase Dispase I Collagenase dispase Dispase in HBSS 1x collagenase I/5x dispase 5U dispase Dispase type II

973 protocols using dispase

Isolation and Purification of Skin Cells

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For isolation of human skin cells 300 µm dermatome sections were incubated in RPMI+10%FCS (BioWest) containing 0.8 mg/ml collagenase (Type IV, Worthington-Biochemical) and 0.05 mg/ml DNase I (Roche) for 12 h. For nanostring analysis and T cell proliferation assay skin was treated with 1 mg/ml dispase (Invitrogen) to separate epidermis and dermis. Dermal DCs were sorted by fluorescence-activated cell sorting (FACS), epidermal LCs were isolated using CD1a microbeads (Miltenyi Biotec) and a magnet (Stemcell techonologies) with a purity of >90%.
For isolation of mouse skin cells, mice were sacrificed and ears were cut off at the base. Ear skin was split into dorsal and ventral halves and incubated in RPMI+10%FCS containing 1 mg/ml dispase (Invitrogen) for 2 h at 37deg. Epidermis and dermis were separated and digested in 0.2 mg/ml collagenase (Type IV, Sigma) for 2 h at 37deg before passing them through a 70 um filter to obtain a single cell suspension.
Mouse skin-draining auricular lymph nodes were isolated, incubated in medium+0.2 mg/ml collagenase for 30 min and passed through 70 um filter.
BHK-21 and C6/36 cells were purchased from the American Type Culture Collection.

Cerny D., Haniffa M., Shin A., Bigliardi P., Tan B.K., Lee B., Poidinger M., Tan E.Y., Ginhoux F, & Fink K. (2014). Selective Susceptibility of Human Skin Antigen Presenting Cells to Productive Dengue Virus Infection. PLoS Pathogens, 10(12), e1004548.

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Isolation of Primary Alveolar Epithelial and Macrophage Cells

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Primary alveolar epithelial cells (AEC) and alveolar macrophages (AM) cells were isolated from TrkA KI mice following the established protocols [51 (link),52 (link)]. After euthanization, mouse lungs were perfused with sterile HBSS (CX30300, Invitrogen), and filled with Dispase (CB40235, Fisher Scientific) followed by 1% low-melt agarose (CX25009, Bioexpress). Lungs were immersed in Dispase for 45 min at room temperature, treated with DNase (AM2238, Invitrogen), mechanically minced into small pieces, and filtered through cell strainers to prepare lung single cell suspension. Cells were incubated with biotinylated anti-CD45 (BD Pharmingen BDB553078) and anti-CD16/CD32 (BD Pharmingen BDB553143) antibody mixture, followed by magnetic separation after addition of BioMag Nuclease-Free Streptavidin particles (Qiagen, 311711). The enriched AEC suspensions were collected and cultured in DMEM/F12 Medium (Lonza, 12001–600) supplemented with 10% FBS on tissue culture-treated dishes.
AMs were harvested from euthanized mice by bronchoalveolar lavage. The pooled lavage fluid was centrifuged for 15 min at 1500 rpm. Cells were re-suspended in DMEM/F12 Medium (Lonza, 12001–600) supplemented with 10% FBS, counted in a hemocytometer, and plated to a cell culture dish for adhesion. The adherent cells were greater than 90% macrophages, as assessed by Diff-Quick staining.

Verma V., Dileepan M., Huang Q., Phan T., Hu W.S., Ly H, & Liang Y. (2022). Influenza A virus activates cellular Tropomyosin receptor kinase A (TrkA) signaling to promote viral replication and lung inflammation. PLoS Pathogens, 18(9), e1010874.

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Isolation of Human Prostate Cells

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Prostate tissues were digested in DMEM/F12/Collagenase/Hyaluronidase/FBS (Invitrogen, Carlsbad, CA) for 3 hours at 37 °C, followed by an additional 1 hour of digestion in 0.25% Trypsin-EDTA (Invitrogen, Carlsbad, CA) on ice. Subsequently, digested cells were suspended in Dispase (Invitrogen, Carlsbad, CA, 5 mg/mL) and DNase I (Roche Applied Science, Indianapolis, IN, 1 mg/mL), and pipetted vigorously to dissociate cell clumps. Dissociated cells were then passed through 70 μm cell strainers (BD Biosciences, San Jose, CA) to get single cells.
Human prostate tissues were chopped into 2–3 mm-long pieces and incubated in 5 mg/ml collagenase type II/ advanced DMEM/F12 (1 ml per 50 mg of prostate tissue) with 10 μM of Y-27632 (STEMCELL technologies) for 5–12 hours. Tissues were pelleted, resuspended, and incubated in chilled 0.25% Trypsin-EDTA for 5 min. Thereafter, human prostate tissues were pelleted, resuspended in Dispase (Invitrogen, Carlsbad, CA, 5 mg/mL) and DNase I (Roche Applied Science, Indianapolis, IN, 1 mg/mL), and pipetted vigorously to dissociate cell clumps. Dissociated cells were then passed through 70 μm cell strainers (BD Biosciences, San Jose, CA) to obtain single cells.

Zhou Z., Jia D., Kwon O., Li S., Sun H., Roudier M.P., Lin D.W., True L., Morrissey C., Creighton C.J., Lee J.K, & Xin L. (2023). Androgen-regulated stromal complement component 7 (C7) suppresses prostate cancer growth. Oncogene, 42(32), 2428-2438.

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Isolation and Culture of Human Epidermal and Corneal Epithelial Cells

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Human epidermal keratinocytes were isolated from foreskin using a well-established protocol described previously^{27 (link)}. In brief, biopsies were washed, minced and digested with dispase [2 U/ml] (Life technologies, Germany) for 18 hours at 4 °C to dissociate the epidermis from the dermis. Thereafter, the epidermis was trypsinized (Thermo Fisher Scientific, Waltham, MA USA) to generate single cell suspensions. The epidermal keratinocytes were cultured in EpiLife® medium containing human keratinocyte growth supplement (Both from Thermo Fisher Scientific, Waltham, MA USA)).
Corneal tissues were transferred and washed in a petri dish with phosphate-buffered saline. Subsequently, the cornea was cut into horizontal stripes of about 2–3 mm and put in a petri dish with dispase [2 U/ml] (Thermo Fisher Scientific, Waltham, MA USA)) for 18 hours at 4 °C. The epithelium was stripped off the central cornea to the limbus with forceps and collected in a new petri dish with fresh phosphate-buffered saline. The epithelial sheets were centrifuged and reduced to small pieces by pipetting for cell seeding. The corneal epithelium was cultured in corneal epithelial cell medium (LGC Standards, Wesel, Germany).

Lotz C., Kiesewetter L., Schmid F.F., Hansmann J., Walles H, & Groeber-Becker F. (2018). Replacing the Draize eye test: Impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation. Scientific Reports, 8, 15049.

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Isolation of Immune Cells from Tissues

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Cells from the intestinal LP were isolated as described previously^{4 (link),44}. Briefly, the tissues were digested with 1 mg/ml Collagenase VIII (Sigma–Aldrich) (SI) or 0.85 mg/ml Collagenase V (Sigma-Aldrich), 1.25 mg/ml Collagenase D (Roche), 1 mg/ml Dispase (Gibco) and 30 μg/ml DNase I (Roche) (colon). Peritoneal cells were isolated by lavage with 4 ml PBS/3% FCS. Mice were perfused with PBS and liver, spleen, lungs and brain were removed and macerated. Lungs were digested with 20 μg/ml Liberase (Roche) and 30 μg/ml DNase (Roche). Spleens and brains were digested with 1 mg/ml Collagenase D (Roche) and 0.15 mg/ml DNase. Liver was digested with 0.5 mg/ml Collagenase A (Roche) followed by Percoll (GE Healthcare) density centrifugation. The ventral and dorsal sheets of the ear were separated from the cartilage and incubated with 2.5 mg/ml Dispase (Gibco) to separate the epidermal and dermal sheets before incubation with 0.25 mg/ml Liberase (Roche) and 0.15 mg/ml DNase. Single cell suspensions were filtered through 100 μm strainers and resuspended in PBS/3% FCS for further analysis.

Guillaume J., Leufgen A., Hager F.T., Pabst O, & Cerovic V. (2023). MHCII expression on gut macrophages supports T cell homeostasis and is regulated by microbiota and ontogeny. Scientific Reports, 13, 1509.

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Nasal Epithelial Cell Isolation Protocol

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HREs were isolated from nasal conchae received from the Clinic for Otorhinolaryngology (RWTH Aachen University Hospital). The local ethics committee approved provision of nasal tissue (EK 067-18). Donors were between 10 and 40 years old with one exception being between 50 and 60 (see supplementary material S6).
Cell isolation was performed using a recently described protocol^{21 (link)}. In short, the tissue was incubated in dispase (2.4 U/mL, Gibco) at 4 °C for 20–22 h. After dispase deactivation with Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% fetal calf serum (FCS, Gibco) and 1% antibiotic/antimycotic (ABM, Gibco), the epithelial cells were scratched from the basal membrane using a cell scraper. Following filtration through a 100 µm cell strainer, the cell solution was centrifuged at 200 g for 5 min. Cells were expanded in T75 cell culture flasks in Airway Epithelial Cell Growth Medium (AECGM, PromoCell) with 0.1% Gentamicin (40 µg/mL, Rotexmedica) in a humidified incubator at 37 °C with 5% CO₂. Exchange of culture medium was performed every 2–3 days.

Luengen A.E., Kniebs C., Buhl E.M., Cornelissen C.G., Schmitz-Rode T., Jockenhoevel S, & Thiebes A.L. (2020). Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro. Scientific Reports, 10, 6963.

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Isolation of Skin T Cells from Different Compartments

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Peripheral mononuclear cells were isolated by Ficoll density gradient centrifugation (density 1.077; GE Healthcare Bio-Sciences, IL, USA). As for isolation of epidermal and dermal T cells, whole-skin specimens were incubated in 5 U/ml dispase (Life Technologies, CA, USA) overnight at 4 °C, and the epidermis was separated from the dermis. The epidermis was cut with scissors and incubated in collagenase III (3 mg/ml; Worthington Biochemical Corporation, NJ, USA) for 90 minutes with deoxyribonuclease (5 jig/ml; Sigma-Aldrich, MO, USA) in RPMI 1640 medium with 10% fetal bovine serum. A single-cell suspension was prepared by pipetting. The dermis was digested in the same way in collagenase III with deoxyribonuclease and further processed by a Medicon tissue disruptor (BD Biosciences, CA, USA). Short-term expansion culture was also applied to collect skin T cells from the whole-skin specimens or the epidermal/dermal specimens separated by dispase (Life Technologies) in the presence of 100 IU/ml of IL-2 (Wako, Osaka, Japan) and 20 ng/ml of IL-15 (Wako). The consistency of the T-cell phenotypes isolated from different body sites or different surgical techniques was also investigated in the subanalyses of our results (Supplementary Fig. 2).

Koguchi-Yoshioka H., Hoffer E., Cheuk S., Matsumura Y., Vo S., Kjellman P., Grema L., Ishitsuka Y., Nakamura Y., Okiyama N., Fujisawa Y., Fujimoto M., Eidsmo L., Clark R.A, & Watanabe R. (2021). Skin T cells maintain their diversity and functionality in the elderly. Communications Biology, 4, 13.

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3D Organoid Culture Protocols

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Organoid RWPE-1 cell cultures were established in 50–60% (v/v) Matrigel membrane matrix (Corning) on non-tissue culture treated plates. MSK-PCa and normal-26Nb cells were maintained as continuous 3D organoid cultures in 75% (v/v) Matrigel growth factor reduced (GFR) membrane matrix (Corning) on non-tissue culture treated plates and harvested from and reseeded in fresh Matrigel every 12–14 days. Fresh cell-type specific medium was added three times per week for all organoid cultures. Organoids were harvested from the Matrigel plug by adding dispase (Life Technologies) at 1 mg/ml (final volume) to the medium, followed by scraping of the well contents with a cell lifter, trituration, incubation for 2 h at 37 °C in a cell culture incubator, and centrifugation at 250×g for 4 min at 4 °C. MSK-PCa and normal-26Nb organoids were then resuspended in TrypLE Express (Life Technologies) supplemented with 1.8 U/ml dispase and incubated at 37 °C with trituration every 5 min until organoids dissociated into a single cell suspension, which was centrifuged again for downstream application or replating. RWPE-1 organoids were treated with 0.05% Trypsin-EDTA for 5 min at 37 °C, which was then neutralized using 2% FBS in PBS, and the organoid cells were centrifuged.

Nath D., Li X., Mondragon C., Post D., Chen M., White J.R., Hryniewicz-Jankowska A., Caza T., Kuznetsov V.A., Hehnly H., Jamaspishvili T., Berman D.M., Zhang F., Kung S.H., Fazli L., Gleave M.E., Bratslavsky G., Pandolfi P.P, & Kotula L. (2019). Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling. Cell Communication and Signaling : CCS, 17, 120.

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Derivation and Culture of hPSCs

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hiPSCs were generated from human fibroblasts as previously described (Kyrkou et al., 2016 (link)), and the H1 hESC line was purchased from Wicell Research Institute (Madison, WI, United States). hPSCs were cultured on six-well tissue culture plates coated with hESC-qualified Matrigel (Corning, 354277) in mTeSR1 medium (StemCell Technologies, 05850) at 37°C and 5% CO₂. Every 4–6 days, cells were passaged enzymatically using 1 mg/ml dispase (Invitrogen, 17105-041) for 2 min at 37°C. hPSC colonies were then harvested, dissociated into small clumps and replated onto Matrigel-coated 6-well plates (ratio 1:6).

Markou M., Kouroupis D., Badounas F., Katsouras A., Kyrkou A., Fotsis T., Murphy C, & Bagli E. (2020). Tissue Engineering Using Vascular Organoids From Human Pluripotent Stem Cell Derived Mural Cell Phenotypes. Frontiers in Bioengineering and Biotechnology, 8, 278.

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Isolation of Lingual Basal Epithelial Cells

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Basal lingual epithelial cells were purified from E16 embryos and P0 newborn mice. Tongues were removed from the mandible and cut to exclude the posterior circumvallate papillae (CV). For RNA and RNA-Seq analysis of control, Krt14-Cre Ring1a^−/−Ring1b^flox/flox mice, E16 tongues were collected. For analysis of control and Krt14-Cre Eed^flox/flox mice, P0 tongues were collected. For taste cell isolation, P0 tongues from P0 TCF/LEF:H2B-GFP(+) mice were collected. Tissues were cut into small pieces and incubated with 1.26U/mL dispase (Invitrogen) and 0.3% type 1 collagenase (Worthington) for one hour at 37°C with 80 rpm shaking. Tissues were washed with 1x PBS, dissociated with 0.25% Trypsin with 2.21mM EDTA (Corning Cellgro; Manassas, VA, USA), and then washed twice with 1xPBS. Cells were stained with 1:400 EpCAM-APC antibodies (Biolegend; San Diego, CA, USA) for 30 min on ice and washed twice with 1x HBSS prior to cell sorting. For ChIP analysis, control, Ring1a/b 2KO, and TCF/LEF:H2B-GFP (+) newborn P0 mice were collected. All cell isolations were performed on a FACS BD Influx or BD FACSAria II instruments (BD, Franklin Lakes, NJ, USA) at the Flow Cytometry Core Facility at Icahn School of Medicine at Mount Sinai.

Bar C., Cohen I., Zhao D., Pothula V., Litskevitch A., Koseki H., Zheng D, & Ezhkova E. (2019). Polycomb Repressive Complex 1 Controls Maintenance of Fungiform Papillae by Repressing Sonic Hedgehog Expression. Cell reports, 28(1), 257-266.e5.

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