Mice at postnatal day 7 (P7) were anesthetized, and cortical slices (400 μm thick) were prepared using a McIlwain tissue chopper (Mickle Laboratory Engineering, Goose Green, UK). Slices are immediately collected and incubated in Accumax (Millipore, Bedford, MA, USA) for 15 min at 37°C. Lysed tissues are homogenized into a single-cell suspension in culture medium by trituration with a flame-polished Pasteur pipette. Cells were resuspended in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12; WelGene, Daegu, Korea) containing 0.9 M sucrose and centrifuged to remove myelin (111 ×g for 10 min). Cells were grown in a 100-mm Petri dish with NSC growth medium (DMEM/F12) containing N-2, B27 supplement without vitamin A (Gibco-Invitrogen, Carlsbad, CA, USA) together with EGF and bFGF (20 ng/ml each; BD Bioscience, San Jose, CA, USA). For Kir4.1-inhibition experiments, cells were treated with 1 μg/ml BaCl2 (Sigma-Aldrich) after 3 h. The size of neurospheres was measured after 1, 3, and 5 d. For differentiation, cells were collected and incubated in Accumax for 5 min. After centrifugation (180 ×g, 3 min), dissociated cells were seeded on plates coated with 0.2 mg/ml poly-L-ornithine and 1 μg/ml fibronectin (Sigma-Aldrich) in the absence of growth factors.
Accumax
Manufactured by Merck Group
Sourced in United States, Germany, Italy
Accumax is a laboratory equipment product designed for precise liquid handling tasks. It functions as an automated pipette, enabling accurate and consistent volumetric liquid transfers. The core purpose of Accumax is to provide a reliable and reproducible liquid handling solution for various applications in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (14 mentions)
Neurobasal medium, by Thermo Fisher Scientific (10 mentions)
Heparin, by Merck Group (8 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Dmem f12, by Thermo Fisher Scientific (6 mentions)
Fc block, by BD (5 mentions)
Penicillin streptomycin, by Lonza (5 mentions)
Fibronectin, by Merck Group (5 mentions)
Neurobasal, by Thermo Fisher Scientific (5 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (5 mentions)
Accuri c6 flow cytometer, by BD (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Facsaria 3, by BD (4 mentions)
Matrigel, by Corning (4 mentions)
40 μm cell strainer, by BD (4 mentions)
Bfgf2, by Thermo Fisher Scientific (4 mentions)
Poly l ornithine, by Merck Group (4 mentions)
Knockout serum, by Thermo Fisher Scientific (4 mentions)
Dnase, by Thermo Fisher Scientific (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
146 protocols using accumax
1
Isolation and Expansion of Neonatal Cortical Neural Stem Cells
2
Isolating Neuronal Cells from Zebrafish Embryos
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After 24 hpf, the NBT dsRed positive embryos were manually sorted. Up to the addition of the deyolking buffer all steps were the same as for early embryos. After the addition of 1 ml deyolking buffer per tube, the embryos were passed 10 times through a 1,000 μl pipet tip, followed by washing twice with deyolking buffer and four times with washing buffer. For better cell dissociation the embryos were rinsed once with Accumax (Millipore) and then resuspended in 1 ml Accumax and transferred to a 15 ml tube. The embryos were incubated at RT for 5 min at the lowest speed of the vortex mixer. The embryos were dissociated by pipetting for 2 min using a 1,000 μl pipet tip, 2 min incubation on the vortex mixer and 1 min of additional pipetting. The cells were spun for 1 min at 300g at RT and washed twice using 1 ml of PBS with 0.5% BSA. 400 μl of PBS with 0.5% BSA was used per tube to resuspend the cells afterwards passed through a 40 μm cell strainer and merged. DNAse I (10 mg/ml in water; Roche) 170 U/ml and 10 mM MgCl2 was added. Cells expressing the DsRed fluorescent protein were FAC sorted with a MoFlo cell sorter (Beckman Coulter GmbH) to obtain a highly enriched fraction for neuronal cells.
3
Flow Cytometry Analysis of Lung Cell Populations
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3D SAEC-F11 VEGFhigh- HMVEC-L and SAEC-F11-HMVEC-L lung aggregates were cultured for 72 h in the presence or absence of rhWnt5a. Aggregates were then dissociated with AccumaxTM (Sigma-Aldrich, St. Louis, USA) solution and washed in PBS once. Single cell suspensions were incubated with Allophycocyanin (APC) conjugated anti-human CD105 (Clone 43A3, BioLegend, San Diego, USA) and Brilliant Violet 421 conjugated anti-human CD31 (Clone VM59, BioLegend, San Diego, USA) for 30min at room temperature in dark. Native lung AC and SCC samples were dissociated by enzymatic digestion (Accumax Solution, Sigma Aldrich, St. Louis, USA) and the single cell suspensions were washed in PBS once, then cells were incubated with APC Cy7 conjugated anti-human CD31 (Clone VM59, BioLegend, San Diego, USA) and APC conjugated CD105 (Clone 43A3, BioLegend, San Diego, USA) antibodies. Cells then were washed in PBS, fixed with 1% PFA and stored at 4 °C in dark until FACS analysis. Labeled cells were analyzed using FACS Canto II flow cytometer (BD Immunocytometry Systems, Erembodegen, Belgium) with BD FACS DIVA software V6 and data were analyzed by FCS Express V3 software.
4
Bone Marrow-Derived Macrophage and Dendritic Cell Generation
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The tibias and femurs of female C57BL/6 mice (aged 8-12 weeks) were isolated and flushed to harvest bone marrow and obtain a progenitor cell population. To generate BMDMs, 4-6×106 bone marrow cells were cultured in non-tissue culture-treated T175 flasks with DMEM/F12 supplemented with 10% (v/v) FBS, 1% (v/v) P/S, 5% (v/v) GlutaMAX and recombinant murine macrophage-colony-stimulating factor (M-CSF, 20 ng/mL). The flasks were supplemented with the additional medium on day three and day six. BMDMs phenotype was confirmed as CD11b+ F4/80+ by flow cytometry. To generate BMDCs, 2x106 bone marrow cells were added to non-tissue culture-treated Petri dishes and cultured in 10 mL of RPMI-1640 supplemented with 10% (v/v) FBS, 1% (v/v) P/S and recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng/mL, Biolegend). Plates were replenished with fresh, supplemented media on day three, six and every other day after that until use. Cell harvesting was performed as follows: BMDMs were detached using AccumaxTM (Sigma), and BMDCs were loosely adherent and could be collected by simple washing. BMDCs phenotype was confirmed as CD11c+ MHCII+ by flow cytometry.
5
Neph3-positive cell isolation from EBs
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Cells were sorted using a BD FACSARIA III (BD Biosciences, San Jose, CA). Single-cell suspensions of EBs in 96-well plates were obtained by collecting all EBs of one 96-well plate in one 15 ml tube, centrifugation at 1500 RPM for 2 min, followed by removal of 14 ml of supernatant. Then, 0.5 ml of Accumax (Chemicon, Germany) was added to the EBs in 0.5 ml of medium and the contents were mixed with a 1 ml pipette and incubated for 15 min at room temperature. For Neph3-positive cells isolation and measurement, EBs were dissociated with Accumax as described above on 11, 13, 15 and 17 DIV and filtered through a cell strainer (BD Biosciences). Isolated cells were incubated in Neph3 monoclonal antibody (30 min, Sheep 1:200, R&D Systems) and labeled with a PE-conjugated secondary antibody (30 min, BD Biosciences). Doublets were excluded from the analysis. Sorted cells were gathered in ice-cold DMEM/F12/N2/10%FCS or NPCs medium and aggregated again in low-cell adhesion 96-well culture plates or 24-well plates.
6
Cell Cycle Analysis of Monolayer and MTS
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Monolayer cells and MTS after six days of culture were dispersed using Accumax (#SCR006 Chemicon) in EMEM medium with 5% fetal bovine serum and filtered through a 70 μm mesh (# 352350, Falcon Corning, Tewksbury, MA, USA). The cell cycle was determined by flow cytometry using the DNA Reagent Kit (Cycletest Plus #340242, BD, Billerica, MA) following the manufacturer’s recommendations. The samples were processed in a BD FACSAria Cell Sorter 3 and analyzed using the ModFit LT 3.2 version software. Each individual assay was performed in triplicate. Statistical comparisons were performed using t-tests.
7
Establishment and Cryopreservation of Tumor Organoids
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Tumor tissues were finely minced and incubated in ACCUMAX™ (Chemicon) for 30 min at 37 °C. One volume of PBS was added to the cell solution, which was then centrifuged at 300 × g for 5 min. Supernatant was removed and cell pellets were resuspended in cold organoid medium (Neurobasal medium (Invitrogen) supplemented with 20 pg/mL EGF (R&D Systems), 10 pg/mL bFGF (Peprotech), 1X B27 (Invitrogen), 2.5% knockout serum replacement, 2.5% fetal bovine serum, 20 mM Glutamax, 1 mM sodium pyruvate, 0.25 µg/mL amphotericin B, and 100 U/mL penicillin-streptomycin). Tumor cell solution was embedded in Matrigel® (growth factor reduced, Corning) at a 1:1.8 ratio of cell solution to Matrigel® solution. A total of 20 µL mixed cell-gel solution was added to six-well plates via 5–7 drops/well and solidified in an incubator (37 °C) for 30–45 min. Organoid medium was added to cover the gel drops; cultures were maintained in a humidified incubator, with 5% CO2, at 37 °C. RB organoids were manually dissociated and passaged at a 1:3 or 1:4 ratio every 3–4 weeks by embedding in fresh Matrigel®. Cold freezing medium (organoid medium containing 10% dimethylsulfoxide) was used to freeze organoids at −80 °C for 24 h prior to long-term storage in liquid nitrogen.
8
Isolation of Mouse Oligodendrocyte Progenitors
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Cortices from neonatal mouse (P1–2) were digested with Accumax (Chemicon, Santa Cruz, CA, USA) for 10 minutes at 37°C. Cortices were triturated and passed through a 70-μm cell strainer (Falcon, USA) to form a single-cell suspension. After centrifugation at 1,000 r/min for 5 minutes, the cells were suspended in DMEM/F12 + 10 % fetal bovine serum (Hyclone, Logan, UT, USA). When the cultured cell mixture reached 65–75 % confluence, the culture medium was replaced with DMEM/F12 + 15 % B104 cell-conditioned medium + 1% N2 supplement + 5 mg/L insulin (Sigma, USA) (modified oligodendrocyte progenitor cell [OPC] growth-medium). The modified OPC growth-medium was replaced daily. On day 7 after incubation with modified OPC growth-medium, mixed glial cells were used for immunostaining.
9
Real-Time Cell Migration Assay
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Cells grown as a monolayer and 6 days MTS were disaggregated with Accumax (Chemicon, MA, USA. #SCR006) were seeded and subjected to real-time migration monitoring using the CIM-Plate 16 and xCELLigence System using a RTCA DP Instrument (ACEA Biosciences, Inc, San Diego CA, 92121 USA). In this system, 40 000 cells of each culture condition were seeded in the upper chamber of a CIM-Plate 16 without FBS. The upper chamber was then placed on the lower chamber of the CIM-Plate 16 containing growth medium supplemented with 10% FBS as an attractant, medium without FBS (negative control). Cell migration was monitored over a period of 24 to 30 hours. MDA-MB-231 cells were used as a positive control (Fig. S1 ). Three assays were performed for each culture condition. Statistical analysis was performed using t-tests.
10
Isolation and Maintenance of hNCSCs
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H9 colonies were dissociated by Accumax (Chemicon, Temecula, CA) and blocked with anti-human Fc- receptor (Miltenyi Biotec, Bergisch Gladbach, Germany). Following Fc blocking, cells were incubated with the phycoerythrin (PE)-conjugated p75 antibody (557196, BD Pharmingen, CA) for 20 min at 4°C. For HNK-1 and p75 double staining, the cells were firstly stained with HNK-1-FITC (322306, Biolegend, CA), then followed by conjugated anti-p75-PE. Compensation for FITC and PE was performed using compensation beads (BD Pharmingen, San Jose, CA). Positive and negative gates were determined using IgG stained and unstained controls.p75+or HNK-1+/p75+hNCSC cells were routinely maintained in self-renewal medium [14 (link)]on 6-well ultra-low attachment plates (Corning, Lowell, MA) at a density of 5×103 cells/ml.
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