Monocytes were harvested directly after isolation and after 24 h stimulation with GM-CSF and adjusted to a final concentration of 1 × 106 cells/mL. Subsequently, 100 µL of cell suspension were seeded per well (in triplicate) and incubated for 2.5 h at 37 °C and 5% CO2 under static conditions using a 96-well culture plate (Greiner-Bio-One, Leipzig, Germany). Supernatants were decanted, and adherent cells were washed with 100 µL PBS per well. Adherent cells were detached by incubation with accutase (100 µL per well) (PAN-Biotech, Aidenbach, Germany) at room temperature for 20 min. Triplicate samples were pooled in a 1.5 mL tube, and the reaction was stopped by adding the cell culture medium to a final volume of 1 mL. Monocytes were then counted using the CASY cell counter.
Accutase
Manufactured by PAN Biotech
Sourced in Germany
Accutase is a gentle, enzymatic cell detachment solution that can be used to dissociate various adherent cell types, including stem cells, without compromising cell viability or altering cell surface markers. It is a proteolytic and collagenolytic enzyme complex that effectively detaches cells from culture vessels.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
96 well culture plate, by Greiner (1 mentions)
Mia paca 2, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Cell scraper, by Sarstedt (1 mentions)
Panc 1, by Merck Group (1 mentions)
Mcf 7, by Merck Group (1 mentions)
Bxpc 3, by Merck Group (1 mentions)
Serological pipettes, by Sarstedt (1 mentions)
Su 86, by Merck Group (1 mentions)
Cytoperm permeabilization buffer plus, by BD (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Nerve growth factor (ngf), by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Poly l ornithine hydrobromide, by Merck Group (1 mentions)
21 protocols using accutase
1
Monocyte Isolation and Stimulation
2
Culturing Cell Lines for Cancer Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All listed cell lines were obtained from American Type Culture Collection. The PDAC cell lines BxPC-3, MiaPaCa-2, PANC-1, SU.86.86, T3M4 and the breast cancer cell line MCF-7 were cultured in RPMI 1640 (MilliporeSigma) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). 293T cells were cultured in DMEM (MilliporeSigma) supplemented with 10% FBS and 1% penicillin-streptomycin. The normal human pancreatic cell line HPDE was cultured in keratinocyte serum-free medium supplemented with 30 µg/ml bovine pituitary extract, 200 pg/ml epidermal growth factor and 1% antibiotic-antimycotic (10,000 U/ml penicillin, 10,000 µg/ml streptomycin and 25 µg/ml Amphotericin B; cat. no. 15240062; Gibco) (all from Thermo Fisher Scientific, Inc.). All cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. Cell culture flasks, dishes, cell scraper and serological pipettes were obtained by Sarstedt AG & Co. KG. Inc. Accutase™ (PAN-Biotech GmbH) was used for detaching cells.
3
MPO Modulates A549 Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 2.5 × 105 A549 cells were seeded in 6-well plates and incubated for up to 24 h at 37 °C. Cells were serum starved for 4 h, followed by treatment with different concentrations of MPO (0.5, 2, 5, 10 µg/mL) for 24, 48 and 72 h. In addition, ddH2O was used as vehicle control. For inhibitor experiments, cells were incubated for 30 min with 10 µM 4-ABAH prior to MPO treatment (5 µg/mL) and incubated for 48 h. A total of 10 µL of 1 mM BrdU-Solution (BD Bioscience, Franklin Lakes, NY, USA; #559619) was added to cells in media 4 h before detaching to allow BrdU to be incorporated into the newly synthesized DNA. Cells were detached from the plate with 300 µL accutase (PanBiotech, Aidenbach, Germany) and collected in 5 mL FACS tubes. The staining was performed without 7-AAD according to the instructions provided by the company. In brief, cells were fixed for 15 min with BD Cytofix/Cytoperm followed by 10 min incubation with BD Cytoperm Permeabilization Buffer Plus. After 5 min incubation with BD Cytofix/Cytoperm, cells were incubated with DNase for 1 h at 37 °C to expose the incorporated BrdU. Anti-BrdU antibody was prepared in a 50 µL 1× Perm Wash and incubated with cells for 20 min at room temperature. Samples were measured using a FACS Canto II flow cytometer (BD Biosciences).
4
Cell Harvesting and Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected 3 days after transfection by trypsinization (Accutase; PAN-Biotech), then washed and resuspended with PBS. FACS analysis of mRFP/BFP/GFP-positive events was performed using a FACSCanto II flow cytometer (BD Biosciences).
5
PC-12 Cell Culture and Signaling Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PC-12 cell line (ATCC CRL-1721) was cultured in RPMI 1640 medium containing L-glutamine (Gibco, Thermofisher, Austria), 5% fetal bovine serum (Gibco, Thermo fisher scientific, Austria), 10% horse serum (Gibco, Thermo fisher scientific, Austria), 100 U/mL penicillin, 100 μg/mL streptomycin (Pan-Biotech, Aidenbach, Germany) at 37°C and 5% CO2 in a humidified incubator. Culture flasks were coated with 250 μg/mL poly-L-ornithine hydrobromide (Sigma-Aldrich, MO, United States). Cultures were passaged using Accutase (Pan-Biotech, Aidenbach, Germany).
For the analysis of TrkA phosphorylation and RhoA/ROCK signaling, PC-12 cells were seeded in 6-well plates (3 × 106 cells/well) for 24 h. To investigate TrkA phosphorylation, PC-12 cells were treated with 10 μM ENDF1 for 5, 15, and 30 min. Treatment with 100 ng/mL NGF (Life Technologies, Carlsbad, Germany) served as positive control to induce TrkA phosphorylation (Kaplan et al., 1991 (link)). Culture medium and 0.01% DMSO were used as negative controls. To investigate impact of ENDF1 on the RhoA/ROCK signaling, PC12 cells were treated with 10 μM ENDF1 for 30 and 120 min. The selective Rho kinase inhibitor Y27632 (stock solution 50 mM in DMSO; Selleck Chemicals, Munich, Germany) was applied at 25 μM for 30 and 120 min as positive control of RhoA/ROCK signaling inhibition. Culture medium and 0.05% DMSO served as negative controls.
For the analysis of TrkA phosphorylation and RhoA/ROCK signaling, PC-12 cells were seeded in 6-well plates (3 × 106 cells/well) for 24 h. To investigate TrkA phosphorylation, PC-12 cells were treated with 10 μM ENDF1 for 5, 15, and 30 min. Treatment with 100 ng/mL NGF (Life Technologies, Carlsbad, Germany) served as positive control to induce TrkA phosphorylation (Kaplan et al., 1991 (link)). Culture medium and 0.01% DMSO were used as negative controls. To investigate impact of ENDF1 on the RhoA/ROCK signaling, PC12 cells were treated with 10 μM ENDF1 for 30 and 120 min. The selective Rho kinase inhibitor Y27632 (stock solution 50 mM in DMSO; Selleck Chemicals, Munich, Germany) was applied at 25 μM for 30 and 120 min as positive control of RhoA/ROCK signaling inhibition. Culture medium and 0.05% DMSO served as negative controls.
6
Phenotyping Immune Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested by Accutase (PAN-Biotech, Aidenbach, Germany), washed with PBS, and incubated with Fc blocking solution (10% human sera, PBS). Cells were stained with a panel of surface markers: PE-conjugated anti-human CD45 with clone HI30 (eBioscience, Frankfrt am Main, Germany); PerCP-Cy5.5-conjugated anti-human CD80 with clone 2D10 (eBioscience, Germany); APCcy7-conjugated anti-human CD206 with clone 19.2 (eBioscience, Germany), and APC-conjugated anti-human CD163 with clone GHI/61 (eBioscience, Germany). SYTOX Blue dead cell staining (Invitrogen, Darmstadt, Germany) was performed to gate out dead cells. Data were acquired on a BD Canto II using BD FACS Diva software. Cell populations were identified on CD45-positive cells on FLowjo (Tree Star, Ashland, OR, USA).
7
Porcine and Seal MDM Infection Kinetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adherent MDM, isolated from porcine or seal blood, were washed twice with PBS and detached using accutase (PAN-Biotech GmbH, Aidenbach, Germany) following the manufacturer’s protocol. The cells were resuspended in RPMI 1640 medium and counted using a Neubauer chamber. MDM were infected with a multiplicity of infection (MOI) of 10:1 by co-incubating 2.5 × 106 bacterial CFU/ml of each S. phocae isolate with 2.5 × 105 MDM cells/ml in RPMI 1640 medium (w/o antibiotics). The experiments were run in triplicates. Subsamples were plated on blood agar plates in triplicates every hour for CFU counting. A control of bacteria was run in RPMI 1640 medium without MDM. Data were tested for statistical significance by using unpaired t-test (p < 0.05) in Prism 9 by GraphPad Software Version 9.0.0 (121).
8
Phenotypic Characterization of BM-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell surface marker expression analyses by flow cytometry were performed to quantify the proportion of the BM-MSC population within the BM-MNC population immediately after BM-MNC isolation and to characterize the isolated BM-MSCs. For this purpose, cells were thawed and expanded until cell culture passage three. Cells were detached with accutase (PAN Biotech), washed twice with PBS and incubated with fluorescently-labeled monoclonal antibodies (all BioLegend) for 15 min at room temperature. Antibodies used (positive markers): CD105 (PerCP cyanine 5.5.), CD73 (brilliant violet 421) and CD90 (phycoerythrin). Antibodies used (DUMP markers): CD14, CD19, CD34, CD45 and HLA-DR (all APC cyanine 7). Following the 15 min of incubation, cells were washed, suspended in FACS buffer (BioLegend) and analyzed by flow cytometry (CytoFLEX LX, Beckman-Coulter). Data analysis was done using Kaluza 2.2 software (Beckman-Coulter).
9
Cell Cycle Analysis of X-Ray Irradiated Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Subcultures of patient fibroblasts were seeded at a density of 4000/cm2 in DMEM with stable glutamine and 15% of FCS (both Sigma, St. Louis, MO, USA) and DNA damage was elicited by exposure of cells to 6 MV X‐ray photons provided by a linear accelerator (Primus series, Siemens Healthcare, Erlangen, Germany) at a dose rate of 3 Gy/min at assay time 0. Matched cultures from treated and untreated fibroblasts were harvested 48 hr postirradiation using Accutase (PAN Biotech, Aidenbach, Germany). For cell cycle analysis, detached cells were pelleted and resuspended at a density of 106/ml in staining buffer containing 4 µg/ml of 4′,6‐diamidino‐2‐phenylindole (DAPI), 100 mM of Tris pH 7.4, 154 mM of NaCl, 1 mM of CaCl2, 0.5 mM of MgCl2, 0.2% of BSA, 0.1% of NP40 in dH2O (Sigma) for a minimum of 30 minutes at 4°C in the dark (Darzynkiewicz, Huang, & Zhao, 2017 ). Bivariate flow histograms were recorded on an analytical, triple laser‐equipped flow cytometer (LSRII, Becton Dickinson, Franklin Lakes, NJ, USA) using UV excitation/emission at 355/460 nm and FACSDiva software for data acquisition. The resulting cell cycle distributions reflecting cellular DNA content and cell cycle progression were quantified by means of the MPLUS AV software package to assess G2 phase accumulation (Phoenix Flow Systems, San Diego, CA, USA) (Schindler & Hoehn, 1999 ).
10
Differentiation of Human Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gibco Dulbecco’s modified Eagle’s medium − high glucose + pyruvate (DMEM), as well as B27 and N2 culture supplements were obtained from Thermo Fisher Scientific (11995-065, 17504-044 and 17502-048, respectively). Fibroblast growth factor-2 (FGF-2) and Epidermal growth factor (EGF) were purchased from Peprotech (100-18B and 315-09). Accutase was obtained from PAN-Biotech (P10-21100). Regarding the substrates, Poly-D-Lysine (PDL) and Laminin were purchased from Thermo Fisher Scientific (A3890401) and Sigma (L2020), respectively. The integrin β1-blocking antibody (Purified Rat Anti-Human CD29) was obtained from BD Pharmingen (552828) and the IgG isotype control (553951) was also purchased from BD Pharmingen.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!