Accumax solution

Manufactured by Merck Group

Sourced in United States

Accumax solution is a laboratory product manufactured by Merck Group. It is designed for the dissociation of adherent cells. The solution contains proteolytic enzymes that facilitate the detachment of cells from surfaces, enabling subsequent cell counting, culturing, or other experimental procedures.

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Accumax (146 citations)

22 protocols using accumax solution

Zebrafish Bone Mineralization Assay

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Six dpf ABxTL zebrafish larvae were injected with uro-I or vehicle (as described above) and 24 h later were euthanized by tricaine-S overdose on ice bath. Bones were harvested as previously described^{57 (link)}. Briefly, soft tissue was removed by incubating larvae with Accumax solution (MilliporeSigma, Catalog#:A7089) under vigorous shaking. Bones were collected using a 70 µm cell strainer, followed by demineralization with 1.2 M HCl. Fluorescent images were captured prior to and after the demineralization step using a Zeiss Axio Imager M2 fluorescence microscope. Porphyrin signal was captured using the red fluorescent channel.
10 mg hydroxyapatite (Acros Organics, Catalog#:1306–06-5) was incubated with 1 mM uro-I, 1 mM copro-I (coproporphyrin-I dihydrochloride, Frontier Scientific, Catalog#:C654-1), vehicle or 0.2% calcein for 30 min in the dark and vortexed every five minutes. Samples were washed and imaged by epifluorescence microscopy as described above.

Bragazzi Cunha J., Elenbaas J.S., Maitra D., Kuo N., Azuero-Dajud R., Ferguson A.C., Griffin M.S., Lentz S.I., Shavit J.A, & Omary M.B. (2021). Acitretin mitigates uroporphyrin-induced bone defects in congenital erythropoietic porphyria models. Scientific Reports, 11, 9601.

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Zebrafish Bone Demineralization and Porphyrin Imaging

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Six dpf ABxTL zebrafish larvae were injected with Uro-I or vehicle (as described above) and 24 h later were euthanized by tricaine-S overdose on ice bath. Bones were harvested as previously described 57 (link) . Briefly, soft tissue was removed by incubating larvae with Accumax solution (MilliporeSigma, Catalog#:A7089) under vigorous shaking. Bones were collected using a 70µm cell strainer, followed by demineralization with 1.2M HCl. Fluorescent images were captured prior to and after the demineralization step using a Zeiss Axio Imager M2 fluorescence microscope. Porphyrin signal was captured using the red fluorescent channel.
10mg hydroxyapatite (Acros Organics, Catalog#:1306-06-5) was incubated with 1mM Uro-I, 1 mM Copro-I (coproporphyrin-I dihydrochloride, Frontier Scientific, Catalog#:C654-1), vehicle or 0.2% calcein for 30min in the dark and vortexed every five minutes. Samples were washed and imaged by epifluorescence microscopy as described above.

Cunha J.B., Elenbaas J.S., Maitra D., Kuo N., Azuero-Dajud R., Ferguson A.C., Rost M.S., Lentz S.I., Shavit J.A., & Omary M.B. (2021). Acitretin mitigates uroporphyrin-induced bone defects in congenital erythropoietic porphyria models.

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Dissociation and Clonogenic Assay of EBs

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Day 14 EBs were dissociated by treatment with Accumax™ Solution (Sigma) at 37 °C followed by mechanical dissociation (as described previously^{27 (link)}). An additional 10-min incubation step was added for the day 21 and day 28 EBs. The resulting cell suspension was filtered and 3 × 10⁴ cells plated in MethoCult^TMH4344 (STEMCELL technologies) semisolid media. On day 14 post-dissociation, colonies were classified and counted based on their morphology.

Buchrieser J., Oliva-Martin M.J., Moore M.D., Long J.C., Cowley S.A., Perez-Simón J.A., James W, & Venero J.L. (2018). RIPK1 is a critical modulator of both tonic and TLR-responsive inflammatory and cell death pathways in human macrophage differentiation. Cell Death & Disease, 9(10), 973.

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Neuroblastoma Cell Response to JQ1

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Each neuroblastoma cell line was washed with 1x Phosphate Buffered Saline (PBS), then lifted and dissociated using Accumax solution (Sigma-Aldrich). The cells were counted using a Cellometer (Nexcelom Bioscience) and 2.8x10⁵ cells of each neuroblastoma cell line were plated into two wells of a 12-well tissue culture plate. Each sample was either treated with 0 (no treatment) or 8µM JQ1, then incubated at 37°C in 5% CO₂. After 48 hours, the plates were examined under bright field conditions using an EVOS M5000 imaging system (ThermoFisher Scientific). Images were examined at a magnification of 40x. The experiment was performed with triplicate independent treatments. Three representative images were acquired from each treatment from which a final representative image was selected.

Mazar J., Gordon C., Naga V, & Westmoreland T.J. (2020). The Killing of Human Neuroblastoma Cells by the Small Molecule JQ1 Occurs in a p53-Dependent Manner. Anti-Cancer Agents in Medicinal Chemistry, 20(13), 1613-1625.

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Dissociation and Colony Assay of Embryoid Bodies

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Twenty-four EBs per condition were harvested using a 100-μm cell strainer, washed in PBS, and EBs were resuspended in 500 μL of Accumax Solution (Sigma). EBs were incubated for 5 min at 37°C, mechanical dissociation was performed by carefully pipetting up and down using a 200 μL pipette for 1 min, after which the EBs were incubated an extra 5 min at 37°C followed by mechanical dissociation for an additional minute. The resulting cell suspension was filtered using a 70 μm cell strainer before being centrifuged for 5 min at 400 × g. Colony-forming cell assay was performed according to the manufacturer's protocol using a cell concentration of 3 × 10⁵ cells/mL resulting in a final plating concentration of 3 × 10⁴ cells per 35 mm dish of MethoCult H4434 (STEMCELL Technologies). Colonies were scored by morphology 14 days after plating.

Buchrieser J., James W, & Moore M.D. (2017). Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages. Stem Cell Reports, 8(2), 334-345.

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Antibody-based Protein Detection Assays

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The antibodies against RagA, phospho-mTOR, mTOR, phospho-p70S6K, p70S6K, and GAPDH were obtained from Cell Signaling Technology (Boston, MA, USA). HRP-conjugated anti-rabbit IgG was purchased from Sigma–Aldrich (St. Louis, MO, USA). NeuN and LAMP2 antibodies were purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG were obtained from Invitrogen (Carlsbad, CA, USA). ChamQ SYBR qPCR master mix was obtained from Vazyme (Nanjing, China). QuantiNova SYBR green PCR kit was obtained from QIAGEN (Valencia, CA, USA). Dulbecco's modified Eagle's medium (DMEM), DMEM/F12, fetal bovine serum (FBS), horse serum (HS), N2, B27, penicillin, streptomycin, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The mammalian expression vector pcDNA3.1 was obtained from Invitrogen (Carlsbad, CA, USA). Puerarin was obtained from Yick-Vic Chemicals & Pharmaceuticals (Hong Kong, China). LPS, Accumax solution, RIPA buffer, and other reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise indicated.

Zhao J., Jia Y., Zhao W., Chen H., Zhang X., Ngo F.Y., Luo D., Song Y., Lao L, & Rong J. (2021). Botanical Drug Puerarin Ameliorates Liposaccharide-Induced Depressive Behaviors in Mice via Inhibiting RagA/mTOR/p70S6K Pathways. Oxidative Medicine and Cellular Longevity, 2021, 7716201.

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Isolation and Culture of Epithelial Cells

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The epithelium was collected from samples digested with 4 U Dispase II solution and then incubated in Accumax solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 28 °C. After tissue dissociation with gentle pipetting, the samples were centrifuged at 1200 rpm for 5 min, and the pelleted cells were used to seed glass coverslips coated with 10 μg/cm² collagen IV (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s modified Eagle medium supplemented with high glucose (Lonza, Basel, Switzerland), 10% FBS, 0.25 U/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), 1% L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% antibiotic/fungizone solution, and 10 ng/mL epidermal growth factor. Images of EP cultures were acquired using a DM2000 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Nikon Digital Sight DS-5Mc camera (Nikon Corporation, Tokyo, Japan).

Piccione M., Facchinello N., Schrenk S., Gasparella M., Pathak S., Ammar R.M., Rabini S., Dalla Valle L, & Di Liddo R. (2021). STW 5 Herbal Preparation Modulates Wnt3a and Claudin 1 Gene Expression in Zebrafish IBS-like Model. Pharmaceuticals, 14(12), 1234.

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Collagen Characterization in Zebrafish Bone

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Adult zebrafish were euthanized with 0.4% tricaine and vertebral bone was dissected manually from two plod2 mutant and two control fish, residual soft tissue was removed by incubation in Accumax solution (Sigma-Aldrich) for 1 hour. Bone was demineralized in 0.1 M HCl at 4°C, washed, and solubilized by heat denaturation in SDS-PAGE sample buffer. The method of Laemmli was used with 6% gels for the denaturant extracts. Collagen α-chains were cut from SDS-PAGE and subjected to in-gel trypsin digestion. Electrospray MS was performed on tryptic peptides using an LTQ XL ion-trap mass spectrometer (Thermo Scientific) equipped with in-line liquid chromatography using a C4 5μm capillary column (300um × 150mm; Higgins Analytical RS-15M3-W045) eluted at 4.μl min. The LC mobile phase consisted of buffer A (0.1% formic acid in MilliQ water) and buffer B (0.1% formic acid in 3:1 acetonitrile:n-propanol v/v). The LC sample stream was introduced into the mass spectrometer by electrospray ionization (ESI) with a spray voltage of 4kV. Proteome Discoverer search software (Thermo Scientific) was used for peptide identification using the NCBI protein database. Proline and lysine modifications were examined manually by scrolling or averaging the full scan over several minutes so that all of the post-translational variations of a given peptide appeared together in the full scan.

Gistelinck C., Witten P.E., Huysseune A., Symoens S., Malfait F., Larionova D., Simoens P., Dierick M., Van Hoorebeke L., De Paepe A., Kwon R.Y., Weis M., Eyre D.R., Willaert A, & Coucke P.J. (2016). Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 31(11), 1930-1942.

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Tumor-Fibroblast Spheroid Formation and Analysis

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Tumour cells and fibroblasts were mixed in a 1:9, 1:4, or 1:2 ratio and seeded into ultralow adherence 96-well U-bottom plates (Corning). Spheroid growth was monitored using the Celigo Image Cytometer (Nexcelom Bioscience), and cell viability was monitored with CellTiter-Glo at the indicated time points. To ensure proper lysis of the spheroids, the incubation time with the CellTiter-Glo reagent was extended from 10 to 30 min before recording luminescence. As indicated cases, spheroids containing D2A1 cells and GFP-fibroblasts were dissociated using Accumax solution (Sigma) and cell numbers were quantified using the Celigo Image Cytometer (Nexcelom Bioscience) and a fluorescent Countess II FL Automated Cell Counter (Invitrogen). For CM-spheroid assays 200 D2A1 cells were seeded in DMEM supplemented with 10% FBS. After 2 days spheres were collected, washed with serum free media and replated in CM supplemented with 2% FBS. Spheroid area was quantified after 8 days culture in CM.

Jungwirth U., van Weverwijk A., Evans R.J., Jenkins L., Vicente D., Alexander J., Gao Q., Haider S., Iravani M, & Isacke C.M. (2021). Impairment of a distinct cancer-associated fibroblast population limits tumour growth and metastasis. Nature Communications, 12, 3516.

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Establishing primary glioblastoma cell cultures

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The histological grade of the surgical specimen from a patient with WHO grade IV glioblastoma was established by histopathological analysis. The tissue sample was collected during surgery and immediately processed. First, the tissue was chopped with a surgical blade in a sterile Petri dish. Chemical dissociation was achieved by adding Accumax solution (Sigma-Aldrich Chemie GmbH) to the chopped tissue for 15 min at room temperature. Dissociated tissue was then centrifuged and resuspended in 5 mL of DMEM/F12 medium, supplemented with 10% FBS, 2 mM L-glutamine, 10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin B solution. DMEM/F12 medium was additionally supplemented with growth factors: 20 µL/mL B-27, 40 ng/mL epidermal growth factor and 20 ng/mL basic fibroblast growth factor, all purchased from Thermo Fisher Scientific. To confirm cell attachment, dissociated tissue was monitored for 48 h before changing the medium. Attached cells were grown until confluence prior to further studies. The glioblastoma origin of the established primary culture, named GBM-6, was confirmed as previously described [25 (link)]. The passage number of GBM-6 was kept low to prevent genetic and epigenetic changes in cells [28 (link)], to ensure the translational potential of obtained results [29 (link)].

Kostić A., Jovanović Stojanov S., Podolski-Renić A., Nešović M., Dragoj M., Nikolić I., Tasić G., Schenone S., Pešić M, & Dinić J. (2021). Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells. Brain Sciences, 11(7), 884.

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