1 × 104 cells were challenged with ADDA5, KCN or NaN3 in the presence or absence of PTM for 3 and 24 hours. Subsequently, wells were loaded with 100 µM of resazurin (Alfa Aesar) that is transformed to fluorescent resorufin by metabolically active cells. The plate was incubated for 2 h at 37 °C. Fluorescence was measured in multimode plate reader (Tecan) at λex 535 nm and λem 590 nm and normalized to untreated control. Four hours after plasma treatment, apoptosis was assessed by staining with cell event™ caspase 3/7 (ThermoFisher) for 30 min at 37 °C. Subsequently, cells were detached using accutase (BioLegend), and accutase containing 4′,6-diamidino-2-phenylindole (DAPI; BioLegend) was added to label terminally dead cells. Cells were subjected to flow cytometric analysis (CytoFlex; Beckman-Coulter). At least 3000 cells were acquired in the caspase−/DAPI− gating region. Data analysis was performed utilizing Kaluza 1.5a software (Beckman-Coulter).
Accutase
Manufactured by BioLegend
Sourced in United States, Germany, France, United Kingdom
Accutase is a cell detachment solution designed for the gentle dissociation and harvesting of adherent cells. It is a mixture of proteolytic and collagenolytic enzymes formulated to effectively detach cells without compromising their viability or altering their functionality.
Automatically generated - may contain errors
Lab products found in correlation
Geltrex, by Thermo Fisher Scientific (6 mentions)
Facscan, by BD (5 mentions)
Cellquest pro, by BD (4 mentions)
Matrigel, by Corning (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Fugene hd, by Promega (2 mentions)
X tremegene hp, by Roche (2 mentions)
Cd31 phycoerythrin pe, by BD (2 mentions)
Mouse anti rat mouse cd90 fitc, by BioLegend (2 mentions)
Hamster anti mouse cd29 fitc, by BioLegend (2 mentions)
Mouse igg fitc, by BD (2 mentions)
Hamster igg fitc, by BioLegend (2 mentions)
Donkey anti sheep igg alexa488 antibody, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (2 mentions)
Kaluza 1.5a software, by Beckman Coulter (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Z fy cho, by MedChemExpress (2 mentions)
Temozolomide, by Santa Cruz Biotechnology (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
97 protocols using accutase
1
Metabolic and Apoptotic Assay Protocol
2
Quantifying Macrophage Phagocytosis of Apoptotic Cells
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CFSE-stained apoptotic and non-apoptotic Jurkat cells were added to MDM plated in 12-well-tissue culture plates, in 10:1 ratio (apoptotic cells/MΦ) for 90 min at 37°C in a 5% CO2 humidified incubator. After co-culture, Jurkat cells were removed, MΦ were washed at least 3 times with phosphate-buffered saline (PBS), detached using AccutaseTM (BioLegends, Paris, France) and incubated with Fc-block (Miltenyi Biotech SAS) in a PBS supplemented with 2% FBS solution. The staining of Jurkat cells with human anti-CD3-PE antibody (BD Biosciences) was used to exclude MΦ with unengulfed lymphocytes bound to their surface. Engulfment efficiency was measured by flow cytometry. EI was calculated according to the following equation: EI = (number of CD3neg CFSEpos MΦ /number of total MΦ) × 100.
3
Xenograft Tumor Growth Monitoring
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All animal experiments were approved by the IACUC of the National Defense Medical Center, Taipei, Taiwan. The SKOV3.PX1_133+44+ cell were collected after dispersing the cell spheres with AccutaseTM (Biolegend) and then washed with DPBS, and centrifuged at 300 g. Suspensions of 1 × 105 or 1 × 104 cells in 0.1 ml DPBS were injected subcutaneously into the right flank of SCID/NOD female mice under aseptic conditions. The tumor size was estimated by the products of the length and width of the subcutaneous tumors. The growth of these tumors was monitored for 3–6 months after injection.
4
CD73 Knockout in Mesenchymal Stem Cells
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iMSCs were transduced with Lenti-U6-sgCD73-SFFV-Cas9-2A-Puro vectors at an MOI of 1 in the presence of 8 μg/ml protamine sulfate. Two days after transduction, cells were treated with 0.5 μg/ml puromycin. At 10–12 days following puromycin selection, cells were dissociated with Accutase and stained with CD73-PE antibody (BioLegend, Inc., San Diego, CA, USA) for 30 min at room temperature. The samples were analyzed on a BD Arial III flow cytometer.
5
Apoptosis Quantification in Cancer Stem Cells
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Post-treatment apoptosis was measured by using the PE-Annexin V Apoptosis Detection Kit (BD Bioscience). Briefly, 1.5 × 105 CSCs from SCC12 cell line were seeded per well, in a 6-well plate for 24 h, and were then treated with SF and/or the chemotherapeutic agents for 72 h. Cells were detached using Accutase (Biolegend, San Diego, California, United States); then, all procedures followed the manufacturer’s protocol. Cells were analysed by flow cytometry using LSR Fortessa (BD Biosciences). Data analysis was performed using FlowJo vX (FlowJo LCC).
6
Transfection and Dye Transfer Assay for Connexin-46
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For GBM CSC transfections, 1×106 cells were plated per well of a 6-well plate adherently on Geltrex (Thermo Fisher Scientific) to obtain a confluence of approximately 75%–80%. Six hours later, cells were transfected with Cx46 or its mutant forms using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, cells were transfected with 5 mg total DNA (4 mg of connexin and 1 mg pEGFP-N3 to track transfection efficiency) using 15 ml FuGENE per well. The following day, cells were removed from the plate using Accutase (BioLegend) and plated for downstream assays. pEGFP-N3 was used as a vector control.
HeLa cells were seeded at 400,000 cells per well in a 6-well plate and transfected using XtremeGene HP (Roche) according to the manufacturer’s protocol. In brief, each well received 2 ug of DNA and 6 uL of XtremeGene reagent. Dye-transfer recipients were plated 24 hours after transfection, and donors were plated and images taken at 48 hours post-transfection. Stable HeLa-Cx46 cells were derived by transfecting HeLa cells with Cx46 (without the GFP tag). Cells were selected with G418 (400 mg/mL), and single-cell clones were tested for the ability to exhibit dye coupling.
HeLa cells were seeded at 400,000 cells per well in a 6-well plate and transfected using XtremeGene HP (Roche) according to the manufacturer’s protocol. In brief, each well received 2 ug of DNA and 6 uL of XtremeGene reagent. Dye-transfer recipients were plated 24 hours after transfection, and donors were plated and images taken at 48 hours post-transfection. Stable HeLa-Cx46 cells were derived by transfecting HeLa cells with Cx46 (without the GFP tag). Cells were selected with G418 (400 mg/mL), and single-cell clones were tested for the ability to exhibit dye coupling.
7
Isolation and Phenotypic Analysis of NLC
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After 12 or 14 days of CLL PBMC culture, floating cells were harvested and NLC were washed twice with PBS and incubated with 1 mL of Accutase (Biolegend, France) for 20 min in 37 °C. Subsequently, 0.5 mL of FBS was added and cells were further detached with gentle scrapping (Sarstedt, USA). Both floating and adherent cells were further combined, washed in PBS, re-suspended in flow cytometry buffer containing 2.5 µg/mL of Human BD Fc Block™ (BD Biosciences, France) and incubated for 15 min at 4 °C. Subsequently, cells were stained with antibodies at saturating concentrations, for 20 min at 4 °C. After washing, cells were re-suspended in PBS, containing 7-AAD (0.8 µg/mL) and immediately analyzed by flow cytometry.
8
Flow Cytometric Characterization of SVF and ad-MVF
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For flow cytometric analyses, isolated SVF single cells and ad-MVF, which were digested in Accutase® (BioLegend, Fell, Germany) for 30 min into single cells, were used. The single cells were analyzed for the expression of the monoclonal rat anti-mouse endothelial cell marker CD31-phycoerythrin (PE) (BD Biosciences, Heidelberg, Germany), the perivascular cell marker mouse anti-α-SMA (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the monoclonal stromal/stem cell surface markers rat anti-mouse CD117-FITC (BD Biosciences), mouse anti-rat/mouse CD90-FITC (BioLegend) and hamster-anti-mouse CD29-FITC (BioLegend). Isotype identical rat IgG-PE or rat IgG-FITC (BD Biosciences), mouse IgG-FITC (BD Biosciences) and hamster IgG-FITC (BioLegend) served as controls. Additionally, cells were analyzed for the expression of the purified polyclonal sheep anti-mouse/human adipocyte marker ASAM (R&D Systems, Wiesbaden, Germany) followed by a secondary donkey anti-sheep IgG-Alexa488 antibody (Molecular Probes, Eugene, OR, USA). Flow cytometric analyses were performed by means of a FACScan (BD Biosciences) and data were assessed using the software package Cell-Quest Pro (BD Biosciences).
9
B2 Binding Assay for MDA-MB-453 Breast Cells
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Breast tumor MDA-MB-453 was lifted from culture dish using Accutase (BioLegend) and incubated with human B2-IgG or B2-Fab in 100 μl PBS and 0.1% BSA for 45 min at 4°C. Bound B2-IgG was stained with goat anti-human IgG-Alexa 568 (1:1000; Invitrogen) for 30 min and analyzed by FACS. B2-Fab was stained with biotinylated goat anti-human IgG (1:1000, Invitrogen) followed by streptavidin-PE staining. In some experiments, cells were pretreated with recombinant heparin lyases III (5 milliunits/ml) for 15 min at RT prior to binding experiments. The MDA-MB-453 cell line was obtained from ATCC (HTB-131). Its identity was authenticated by SRT profiling, and it was tested negative for mycoplasma in the lab.
10
Cardiac Myoblast Cell Line H9c2 Colchicine Assay
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Rattus norvegicus, cardiac myoblast cell line H9c2 was a gift from Dr. Jin O-Uchi. Cells were maintained in Dulbecco’s modified Eagle’s medium with 4500 g/L glucose (DMEM) (Thermo Scientific), supplemented with 10% fetal bovine serum (Equitech Bio, Kerrville, TX, USA), 100 Units/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were passaged with Accutase (Biolegend, San Diego, CA, USA). To examine the effects of colchicine, 200,000 cells were plated per well of a 48-well dish with or without 100 nM colchicine. The following day, protein was collected in Laemmli sample buffer supplemented with HALT protease and phosphatase inhibitor cocktail (ThermoFisher).
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