Stempro accutase

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Italy, Canada, Sweden

StemPro Accutase is a cell dissociation reagent used for the gentle dissociation of adherent cells, including stem cells and other sensitive cell types. It is a non-enzymatic, animal-component-free solution designed to maintain cell viability and preserve cell surface epitopes.

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Close spelling variants of this product

StemPro Accutase (A11105-01) StemPro Accutase Cell Dissociation Reagent Stempro Accutase Cell Dissociateion Reagent Accutase

352 protocols using stempro accutase

Evaluating Mammosphere Formation Capacity

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MCF10A or MCF10DCIS cells were grown to 50–60% confluence and cells were detached with StemPro Accutase (Life Technologies). Cells were then plated at 10,000 cells/mL in 6-well ultra-low attachment plates and maintained in Mammocult serum-free medium supplemented with hydrocortisone and heparin (Stem Cell Technologies). Cells were treated with 1α25(OH)₂D₃ (100 nM) or BXL0124 (10 nM). For secondary and tertiary mammosphere culture, primary mammospheres were collected and enzymatically dissociated using StemPro Accutase (Life Technologies). Then, cells were re-plated at a density of 5,000 cells/mL for subsequent passages. Photos of mammospheres were taken, and the number of mammospheres was counted to determine the mammosphere forming efficiency (MFE). The MFE was calculated by dividing the number of mammospheres (≥100 μm) formed by the number of single cells seeded. Roundness of spheres was obtained by analysis of photos with ImageJ software (US National Institutes of Health, Bethesda, MD). The formula used to calculate roundness is 4 × ([area]/π[major axis]²). A value of 1.0 represents an object that is perfectly round. A value of 0.0 represents an object that is formless. Experiments were repeated in triplicates.

Wahler J., So J.Y., Cheng L.C., Maehr H., Uskokovic M, & Suh N. (2014). Vitamin D Compounds Reduce Mammosphere Formation and Decrease Expression of Putative Stem Cell Markers in Breast Cancer. The Journal of steroid biochemistry and molecular biology, 148, 148-155.

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Mammosphere Formation Assay for SUM159 Cells

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SUM159 cells were grown to 50-60% confluence and cells were detached with StemPro Accutase (Life Technologies, CA). Cells were then plated at 2,000 cells/mL in 6-well ultra-low attachment plates and maintained in Mammocult serum-free medium supplemented with hydrocortisone and heparin (Stem Cell Technologies, Vancouver, Canada). Cells were treated with 1α,25(OH)₂D₃ or BXL0124 for five days for each passage. For secondary and tertiary mammosphere culture, primary mammospheres were collected and enzymatically dissociated using StemPro Accutase (Life Technologies, CA). Then, cells were re-plated at a density of 2,000 cells/mL for subsequent passages. Images of mammospheres were taken, and the number of mammospheres was counted to determine the mammosphere forming efficiency (MFE). The MFE was calculated by dividing the number of mammospheres (≥100 μm) formed by the number of single cells seeded. Experiments were repeated three times.

Shan N.L., Wahler J., Lee H.J., Bak M.J., Gupta S.D., Maehr H, & Suh N. (2016). Vitamin D compounds inhibit cancer stem-like cells and induce differentiation in triple negative breast cancer. The Journal of steroid biochemistry and molecular biology, 173, 122-129.

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Modulating Retinoic Acid Receptor Pathway

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For RAR pathway modulation, cells were treated for 24 hours either with 0.5 µM RA or with 5 µM BMS493 (a pan‐retinoic acid receptor inverse agonist) or with a combination of RA and BMS493, directly diluted in the culture media. For EBs formation assay, cells were dissociated into single cells using StemPro Accutase (Thermo Fisher Scientific) and cultured for 7 days on poly (2‐hydroxyethyl methacrylate) (Sigma‐Aldrich) – coated dishes in mTeSR1 medium supplemented with 10 µM of the Rho‐kinase inhibitor Y‐27632 (Selleckchem) for the first three days. At day 7, floating EBs were transferred on 5 µg/mL Biolaminin 521LN (Biolamina)‐coated plates and cultured in adhesion for additional thirteen days in medium consisting of DMEM/F12 containing 20% knockout serum replacement (KSR, Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% non‐essential Amino Acids (Thermo Fisher Scientific), 100 µM 2‐mercaptoethanol and 0.5% penicillin and streptomycin.

Parrotta E.I., Scalise S., Taverna D., De Angelis M.T., Sarro G., Gaspari M., Santamaria G, & Cuda G. (2019). Comprehensive proteogenomic analysis of human embryonic and induced pluripotent stem cells. Journal of Cellular and Molecular Medicine, 23(8), 5440-5453.

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Metabolomics Profiling of iPSCs

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The generation of the PWB and control iPSC lines from skin biopsies are detailed in our recent study [16 (link)]; these cell lines were maintained and propagated under feeder-free conditions using an Essential 8 Medium on a geltrex-coated plate (ThermoFisher, Waltham, MA, USA). The following iPSC lines were used in this study: PWB_4221_3, PWB_4221_6, PWB_3921_9, PWB_3921_16, control_52521_8, and control_52521_9 [16 (link)]. There were experimental duplications for PWB_4221_3, PWB_3921_9, and control_52521_8, resulting in a total of 9 samples. The iPSCs (~10⁶ cells) were dissociated using StemPro Accutase (ThermoFisher, Waltham, MA, USA); cell pellets were collected and stored at −80 °C until processing. Samples were thawed with the addition of 300 µL of 80% methanol. Samples were vortexed for 60 s, sonicated for 30 min at 4 °C, and incubated at −20 °C for one hour. The samples were then subjected to centrifugation at 12,000 rpm at 4 °C for 15 min. The supernatant (200 µL) was mixed with 5 µL of L-o-Chlorophenylalanine (0.14 mg/mL) for liquid chromatography–mass spectrometry (LC–MS) analysis.

Nguyen V., Kravitz J., Gao C., Hochman M.L., Meng D., Chen D., Wang Y., Jegga A.G., Nelson J.S, & Tan W. (2023). Perturbations of Glutathione and Sphingosine Metabolites in Port Wine Birthmark Patient-Derived Induced Pluripotent Stem Cells. Metabolites, 13(9), 983.

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Neurosphere Culture from Amniotic Fluid

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Amniotic fluid was harvested as described for AF-MSC. Using a modification of previously described procedures, AF was placed into culture flasks containing NSC medium composed of Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, ThermoFisher), supplemented with 4% chicken embryo extract (Gemini Bio-Products, West Sacramento, CA), 2% PSA, 1X N-2 supplement (ThermoFisher), 20ng/ml recombinant fibroblast growth factor basic (FGF, R&D Systems, Minneapolis, MN), and 20 ng/ml recombinant rat epidermal growth factor (EGF, R&D Systems).^{12 (link),13 (link)} Medium was changed every four days, during which time NSC aggregates known as neurospheres formed. After two weeks of growth, these neurospheres were then dissociated to individual NSC using a combination of chemical dissociation with StemPro Accutase (ThermoFisher) and mechanical dissociation via gentle pipetting.

McCulloh C.J., Olson J.K., Wang Y., Vu J., Gartner S, & Besner G.E. (2017). Evaluating the Efficacy of Different Types of Stem Cells in Preserving Gut Barrier Function in NEC. The Journal of surgical research, 214, 278-285.

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Evaluating Melanoma-Initiating Cells' Self-Renewal

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The formation of tumorspheres was used to evaluate the self-renewal ability of melanoma-initiating cells. In brief, melanoma cells were suspended in a stem cell medium and plated at a density of 5,000 cells/well in Corning^® ultralow adherent six-well dishes (Corning Incorporated, Corning, NY, USA). The stem cell medium contained DMEM/F12, B27 supplement (1×), ITS (1×), EGF, and bFGF (both at a concentration of 20 ng/mL). The cells were cultured in the stem cell medium for 7 days, and then tumorspheres were counted under a conventional microscope. For second passage tumorspheres, those of the first passage were washed with PBS, dissociated with a cell dissociation reagent (StemPro^® Accutase^®; Thermo Fisher Scientific, Waltham, MA, USA), and then propagated.

Chen X., Zhang Z., Yang S., Chen H., Wang D, & Li J. (2018). All-trans retinoic acid-encapsulated, CD20 antibody-conjugated poly(lactic-co-glycolic acid) nanoparticles effectively target and eliminate melanoma-initiating cells in vitro. OncoTargets and therapy, 11, 6177-6187.

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Macrophage Activation by Tumor Secretome

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Previously obtained M0 and M1 macrophages were cultured in a total volume
of 2 ml of RPMI-S containing 30% of the supernatant of HeLa, SiHa, or
C-33A cell lines for 1 h; subsequently, the cells were harvested with
Accutase (StemPro^® Accutase^®; Thermo Fisher
Scientific, Waltham, MA, USA) and stained for assessment of the
phosphorylation of transcription factors (STAT1, STAT6, and NF-κB-p65)
by Flow Cytometry (FC).
For assessment of cytokines and growth factors, M0 macrophages were
cultured in RPMI-S containing 30% of the supernatants of HeLa, SiHa,
or C-33A cell lines or for 3 and 5 d at 37°C in a humidified
atmosphere with 5% of CO₂. We used M1 and M2 macrophages
like experimental controls of this assays. Afterward, the supernatant
was collected under sterile conditions, centrifuged at
400 g for 10 min, and stored at –80°C until
determination of cytokines and growth factors.

Sánchez-Reyes K., Pedraza-Brindis E.J., Hernández-Flores G., Bravo-Cuellar A., López-López B.A., Rosas-González V.C, & Ortiz-Lazareno P.C. (2019). The supernatant of cervical carcinoma cells lines induces a decrease in phosphorylation of STAT-1 and NF-κB transcription factors associated with changes in profiles of cytokines and growth factors in macrophages derived from U937 cells. Innate Immunity, 25(6), 344-355.

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Neurosphere Formation Assay with ZIKV

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NSCs were dissociated using StemPro Accutase (Thermo Fisher Scientific, #A1110501), following manufacturer’s instruction. Single cell suspensions were obtained using 40 μm cell strainers (Stemcell Technologies, #27305). 1×10⁵ NSCs were seeded in 6 well plates, treated with ZIKV or lentiviruses within 24 h after seeding, and the numbers of neurospheres (>30 μm) formed after 7 days of in vitro culture were counted for each condition, using an inverted light microscope (Keyence BZ-9000), as previously reported (Cloëtta et al., 2013 (link); Keyoung et al., 2001 (link); Wang et al., 2013 (link)).

Zeng J., Dong S., Luo Z., Xie X., Fu B., Li P., Liu C., Yang X., Chen Y., Wang X., Liu Z., Wu J., Yan Y., Wang F., Chen J.F., Zhang J., Long G., Goldman S.A., Li S., Zhao Z, & Liang Q. (2020). The Zika Virus Capsid Disrupts Corticogenesis by Suppressing Dicer Activity and miRNA Biogenesis. Cell stem cell, 27(4), 618-632.e9.

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CRISPR/Cas9 Genome Editing of CCM1 in iPSCs

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The CRISPR/Cas9 genome editing protocol to generate the CCM1^−/− iPSC lines used in this study was already described before [20 (link)]. In brief, AICS-0023 iPSCs were transfected with single guide RNA (sgRNA):Cas9 ribonucleoprotein complexes with the target region located in exon 10 of CCM1 (LRG_650t1, 5′-GGAGCTCCTAGACCAAAGTA-3′; Integrated DNA Technologies, Coralville, IA, USA) using Lipofectamine Stem Transfection Reagent (Thermo Fisher Scientific). IPSCs were reverse transfected following detachment with StemPro Accutase (Thermo Fisher Scientific) on growth factor reduced Matrigel-coated 24-well plates. For single-cell cloning, statistically 0.5 cells/well were plated on 96-well plates in Essential 8 Flex medium supplemented with CloneR (STEMCELL Technologies, Vancouver, BC, Canada). Genomic DNA of expanded cells was isolated and sequenced by Sanger sequencing.

Pilz R.A., Skowronek D., Mellinger L., Bekeschus S., Felbor U, & Rath M. (2023). Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature. International Journal of Molecular Sciences, 24(4), 3993.

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Islet Cell Dissociation and shRNA Knockdown

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Islets were dissociated with StemPro Accutase (Thermo) and seeded into AggreWell plates in the presence of pLKO.5-encoded lentiviruses producing either nontargeting control shRNA or a pool of lentiviruses producing Nr4a1 shRNA (Supplemental Table 7). Aggregated islet cells were harvested 3 or 4 days later for RNA or perifusion assays, respectively.

Wortham M., Liu F., Harrington A.R., Fleischman J.Y., Wallace M., Mulas F., Mallick M., Vinckier N.K., Cross B.R., Chiou J., Patel N.A., Sui Y., McGrail C., Jun Y., Wang G., Jhala U.S., Schüle R., Shirihai O.S., Huising M.O., Gaulton K.J., Metallo C.M, & Sander M. (2023). Nutrient regulation of the islet epigenome controls adaptive insulin secretion. The Journal of Clinical Investigation, 133(8), e165208.

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