Accutase cell detachment solution

Manufactured by BioLegend

Sourced in United States

Accutase cell detachment solution is a non-enzymatic, animal-free reagent used for the gentle harvesting and dissociation of adherent cells from cell culture surfaces.

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12 protocols using accutase cell detachment solution

Umbilical Cord-Derived Mesenchymal Stem Cell Isolation

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The umbilical cord used in our study was collected after caesarean section, washed with phosphate-buffered saline (PBS) solution supplemented with antibiotic-antimycotic solution and cut into small (5 mm) slices. The explants of umbilical cord were put onto the plastic flask and were cultured with special growth medium for mesenchymal stem cells (DMEM Low Glucose, Biowest, Riverside, MO, USA), supplemented with platelet lysate in a standard culture conditions under 21% of O₂ and 5% of CO₂ at 37 °C for 7 days. After this time, the explants were removed, and the cells were passaged using Accutase cell detachment solution (BioLegend, San Diego, CA, USA). After reaching the appropriate number of cells, the cells were cryopreserved in a freezing medium (BI Biological Industries, Cromwell, CT, USA) for further experiments.
The CardioCell was manufactured in a licensed GMP (good manufacturing practice)-compliant facility, ensuring that products were consistently produced and controlled according to quality standards. The umbilical cords were obtained as part of the project STRATEGMED II (Agreement number Strategmed2/265761/10/NCBR/2015).
An inverted light microscope Olympus IX70 with phase contrast optics, equipped with Canon EOS1100D digital photo camera, was used for routine observation, analysis and documentation of the MSCs at a morphological level.

Musiał-Wysocka A., Kot M., Sułkowski M, & Majka M. (2019). Regenerative Potential of the Product “CardioCell” Derived from the Wharton’s Jelly Mesenchymal Stem Cells for Treating Hindlimb Ischemia. International Journal of Molecular Sciences, 20(18), 4632.

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Kinetics of Virus-Like Particle Endocytosis

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VLPs were stained with DiD (Thermo Fisher Scientific, Eugene, OR) at 20 µg/mL for 30 min at 22 °C, and then purified from free dye using gel filtration columns (PD MiniTrap G-25, GE Healthcare, Buckinghamshire, UK). MDMs were detached from plastic plate surface using Accutase Cell Detachment Solution (BioLegend, San Diego, CA) and plated on 96-well Nunclon Delta black flat-bottom plates (Thermo Fisher Scientific, Roskilde, Denmark) at 5 × 10⁴ cells/well. The following day, MDMs were exposed to DiD-labeled VLPs (HA concentration 15.0 µg/mL) at 4 °C for 1 h. Endocytosis inhibitors were applied in ice-cold medium supplemented with 10% FBS: dynasore hydrate 50 µM, genistein 200 µM, amiloride hydrochloride 1 mM, cytochalasin D 4 µM (all from Sigma-Aldrich, St. Louis, MO). DiD fluorescence was measured with pre-heated (37 °C) spectrophotometer (Infinite 200 PRO, Tecan, Männedorf, Switzerland) at 15-min intervals over 2 h. Fusion efficiency was determined following the addition of Triton X-100 (Sigma-Aldrich) to each well (final concentration 1%) to obtain full DiD dequenching.

Makarkov A.I., Golizeh M., Ruiz-Lancheros E., Gopal A.A., Costas-Cancelas I.N., Chierzi S., Pillet S., Charland N., Landry N., Rouiller I., Wiseman P.W., Ndao M, & Ward B.J. (2019). Plant-derived virus-like particle vaccines drive cross-presentation of influenza A hemagglutinin peptides by human monocyte-derived macrophages. NPJ Vaccines, 4, 17.

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Isolation and Culture of WJMSCs

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The umbilical cords were collected after Caesarean sections. Written consents were obtained from parents. The umbilical cords were washed with phosphate‐buffered saline supplemented with antibiotic‐antimycotic solution, cut into small explants and plated into a plastic flask. Explants were cultured with a growth medium for MSCs (DMEM Low Glucose, Biowest), supplemented with the platelet lysate in standard culture conditions under 21% of O₂ and 5% of CO₂ at 37°C. Next, the explants were removed, and the cells were passaged using the Accutase cell detachment solution (BioLegend). After reaching the appropriate number of cells, WJMSCs were used for further experiments.

Krzyżak A.T., Habina‐Skrzyniarz I., Mazur W., Sułkowski M., Kot M, & Majka M. (2022). Nuclear magnetic resonance footprint of Wharton Jelly mesenchymal stem cells death mechanisms and distinctive in‐cell biophysical properties in vitro. Journal of Cellular and Molecular Medicine, 26(5), 1501-1514.

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Differentiation of Bone Marrow-Derived Macrophages

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Bone marrow–derived macrophages (BMMs) were differentiated from stem cells obtained from the femur and tibia of a naive C57BL/6 mice. Briefly, the bones were separated from the surrounding muscles and the content of the bones was flushed with 5 ml of RPMI 1640 into a polypropylene petri dish using a syringe and a 25-G needle and made into single-cell suspensions. Following depletion of RBCs with ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA [pH 7.2–7.4]), cells were seeded at a density of 25 × 10⁶/ml in T75 ml flasks in complete RPMI 1640 medium supplemented with 10% FBS (VWR Seradigm), 2 mM GlutaMAX (Gibco), 1 mM sodium pyruvate (Corning Cat #25-000-CI), 10 mM HEPES (Sigma-Aldrich) and β-Mercaptoethanol with 25ng/mL M-CSF (Biolegend Cat# 574808) for 72 h at 37 °C in a CO₂ incubator. At 72 h adhered cells reached confluency of 80-90% and non-adhered cells were washed off with PBS. BMMs were detached using Accutase® Cell Detachment Solution (Biolegend Cat# 423201) for 15 min at 37 °C in a CO₂ incubator. Detached BMMS were infected with distinct parasite strains at a cell/parasite ratio of 1:10. After a 6-h incubation (at 37 °C), the cells were washed twice (centrifuged at 600 rpm for 5 min) to remove free parasites.

Zayats R., Mou Z., Yazdanpanah A., Gupta G., Lopez P., Nayar D., Koh W.H., Uzonna J.E, & Murooka T.T. (2023). Antigen recognition reinforces regulatory T cell mediated Leishmania major persistence. Nature Communications, 14, 8449.

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Adherent Culturing of Neurospheres

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To transition these primary NPA GFP⁺ neurospheres to an adherent phenotype, T25 flasks were first coated with 15 µg laminin in 3 ml of DPBS, enriched with CaCl₂ and MgCl₂ (Sigma, D8662‐500ML). Flasks were then incubated for 2 h at 37 °C under 5% CO₂ conditions. Following this, the coated flasks were incubated at 4 °C to solidify overnight. Prior to introducing the glioma cells, it is essential to rinse the flasks twice with cold DPBS enriched with CaCl₂ and MgCl₂ to remove unbound laminin followed by one washing with DPBS without CaCl₂ and MgCl₂. NPA GFP⁺ neurospheres were then treated with Accutase cell detachment solution (Biolegend, 423 201) for 2 min to make a single cell suspension. This single cell suspension was then cultured on the laminin coated T25 flasks using DMEM medium containing 10% FBS. Notably, the cells underwent at least 5 passages before setting up any time‐lapse experiments was ensured. After these passages, we observed that the NPA GFP⁺ cells had begun to show collective organization patterns, particularly structures resembling oncostreams.

Faisal S.M., Clewner J.E., Stack B., Varela M.L., Comba A., Abbud G., Motsch S., Castro M.G, & Lowenstein P.R. (2024). Spatiotemporal Insights into Glioma Oncostream Dynamics: Unraveling Formation, Stability, and Disassembly Pathways. Advanced Science, 11(18), 2309796.

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Murine Melanoma Tumor Extraction and Analysis

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Murine melanoma cell lines B16F10 (CRL-6475; ATCC) were cultured in RPMI 1640 (2 mM glutamine, 100 μg/mL streptomycin, 10% FBS, 100 U/mL penicillin, and 1 M HEPES). Prior to the use, the cells with 70-80% confluence were detached with trypsin-EDTA (0.25%) and washed with PBS twice. For the melanoma models, C57BL/6 mice were subcutaneously (s.c.) inoculated with 1 × 10⁶ cells of B16F10. After 7 days of tumor-cell injection, the mice were further divided in three groups and s.c. injected with PBS, FimH (2.5 mg/kg), or LPS (1 mg/kg) at the distance of the tumor (21 (link)). Total tumors were excised in a sterile dish containing 5 mL of RPMI-1640 media at room temperature (RT) and minced into small pieces using fine scissors. After filtering through a 70-μm cell strainer, the cell suspension was mixed with 10 mL of Ficoll-Paque media and then gently layered on 10 mL of fresh Ficoll-Paque media and centrifuged (1025 × g, 20 min, 20°C) with slow acceleration and the brakes turned off (23 (link)). The lymphomononuclear layer was collected and cultured for 40 min at 37°C. After three washes, the adherent cells remaining in the plate were detached by using Accutase^® Cell Detachment Solution (BioLegend, San Diego, California, USA) and washed for further analysis.

Zhang W., Xu L., Zhang X., Xu J, & Jin J.O. (2023). Escherichia coli adhesion portion FimH polarizes M2 macrophages to M1 macrophages in tumor microenvironment via toll-like receptor 4. Frontiers in Immunology, 14, 1213467.

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Quantifying Cell Surface Sialylation

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To correlate changes in ST6Gal-I overexpression with alterations in cell surface α2,6 protein sialylation, cells were stained with the SNA lectin, which specifically binds α2,6 sialic acids. Accutase cell detachment solution (Biolegend; 423201) was used to suspend cells. The cells were first fixed for 20 min in 2% paraformaldehyde at RT. Following two washes with FACS buffer (1× PBS without Ca²⁺ and Mg²⁺, 5 mM EDTA, 1% BSA, 25 mM Hepes, 0.02% sodium azide), the cells were incubated for 30 min at RT with a 1:200 dilution of SNA Lectin conjugated to Cy5 (Novus Biologicals, CL-1305-NB). The cells were spun down and washed with FACS buffer and transferred to a flow tube. The BD LSR II flow cytometer (BD Biosciences) was used to analyze surface staining. Mean fluorescence intensity values were calculated using FlowJo. To confirm changes in cell surface EGFR, the cells were stained with 1:40 dilution of PE Anti-EGFR antibody (Abcam; ab130738). All other treatment steps were consistent.

Rao T.C., Beggs R.R., Ankenbauer K.E., Hwang J., Ma V.P., Salaita K., Bellis S.L, & Mattheyses A.L. (2022). ST6Gal-I–mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion. The Journal of Biological Chemistry, 298(4), 101726.

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Isolation of Pancreatic Islets

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The pancreas was perfused through the bile duct with 250 μg/ml collagenase P (Sigma Aldrich, St. Louis, MO, USA) diluted in HBSS (Sigma Aldrich). After perfusion the pancreata were removed, cut into small pieces digested with collagenase P at 37°C for 15 minutes. After vigorous shaking for 1 min, the homogenous mixture was passed through a metal sieve and larger chunks were further broken down through grinding. Subsequently, the mixture was poured through a 70 μm cell filter allowing exocrine cells passing through, whereas islets remained retained in the strainer. The islets were transferred into Petri dishes, handpicked under a microscope and incubated 10 min at 37°C with Accutase^® cell detachment solution (Biolegend, San Diego, CA, USA) to obtain a single cell solution. The exocrine pancreas was separately incubated with DNase I (20 U/ml) (Life Technologies, Carlsbad, CA, USA) at 37°C for 1 hour. The obtained cells were passed through a 100 μm filter and resuspended in NH₄CL red cell lysis buffer for 5 min to eliminate contaminating erythrocytes.

Ma Z, & Ruedl C. (2022). Turnover Kinetics of Pancreatic Macrophages in Lean and Obese Diabetic Mice. Frontiers in Endocrinology, 13, 858422.

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Cell Viability Evaluation by Trypan Blue

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Cell viability was estimated by trypan blue exclusion assay. Cells were released with Accutase^® Cell Detachment Solution (BioLegend), and cell counting was performed using the Countess Automated Cell Counter (Invitrogen).

Bueno M.R., Ishikawa K.H., Almeida-Santos G., Ando-Suguimoto E.S., Shimabukuro N., Kawamoto D, & Mayer M.P. (2022). Lactobacilli Attenuate the Effect of Aggregatibacter actinomycetemcomitans Infection in Gingival Epithelial Cells. Frontiers in Microbiology, 13, 846192.

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Characterizing cKit+ Embryonic Fibroblasts

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Anti-mouse cKit (clone 2B8) APC (Cat# 553356) (BD Biosciences, San Jose, CA), and anti-mouse CD3 antibody (clone 17A2) APC (Cat#100235) (Biolegend, San Diego, CA) were used for flow cytometry. For binding analyses of cKit aptamer-miR-26a chimera, the miR-26a was conjugated with Alexa Floure (AF) 488-green fluorescent dye (Integrated DNA Technologies). The cKit receptor^+/- mouse embryonic fibroblast cell line (MEF) was collected with Accutase cell detachment solution (Biolegend) and incubated with 1 μM miR-26a chimera for 10 min in PBS buffer containing 0.45% glucose, 100 mg/L tRNA, 0.1% BSA, 2.5 mM MgCl₂. For inflammatory cytokine analyses, the levels of IL-6, TNF-α and IFN-γ in peripheral blood were determined by cytometric beads assay kit for mouse inflammation (BD Bioscience). These flow cytometry analyses were performed using FACS Canto II (BD Bioscience) and the data were analyzed by FlowJo software (FLOWJO, Ashland, OR).

Tanno T., Zhang P., Bailey C., Wang Y., Ittiprasert W., Devenport M., Zheng P, & Liu Y. (2022). A novel aptamer-based small RNA delivery platform and its application to cancer therapy. Genes & Diseases, 10(3), 1075-1089.

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