At enrollment whole blood (10 ml) was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson & Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMC) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA, USA) and washed twice in PBS at 1500 RPM for 10 min; viable leukocytes were determined using a Scepter 2.0 Handheld Automated Cell Counter (Millipore, Billerica, MA, USA).
Scepter 2.0 handheld automated cell counter
Manufactured by Merck Group
Sourced in United States
The Scepter 2.0 Handheld Automated Cell Counter is a compact and portable device designed for accurate cell counting. It utilizes advanced technology to provide reliable cell concentration and viability measurements.
Automatically generated - may contain errors
Lab products found in correlation
Lympholyte separation medium, by Cedarlane (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Elx800, by Agilent Technologies (1 mentions)
Wst 1 cell proliferation reagent kit, by Merck Group (1 mentions)
Transwell plate, by Corning (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
96 well culture plate, by Corning (1 mentions)
Multidrop combi, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
El 800 spectrophotometer, by Agilent Technologies (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Polystyrene microspheres, by Bangs Laboratories (1 mentions)
Facsaria special order research product flow cytometer sorter, by BD (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
29 protocols using scepter 2.0 handheld automated cell counter
1
Peripheral Blood Mononuclear Cell Isolation
2
Cell Proliferation Assay for TQ Treatment
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The effect of TQ on cell proliferation was analyzed by a colorimetric cell proliferation assay using WST-1 Cell Proliferation Reagent Kit (Sigma-Aldrich, catalog no. 11644807001). Briefly, the cells were seeded in 96-multiwell plates at a density of 104 cells/well (counted using Scepter 2.0 Handheld Automated Cell Counter, Millipore, Billerica, MA, USA; catalog no. PHCC20040) for MDA-MB-468 cells or 4 × 104/well for JK cells. After 24 h of incubation, the cells were exposed to different concentrations of TQ for the desired times. Cell proliferation rate was evaluated through a rapid WST-1 reagent. After incubation for the above-mentioned time, 10 µL of the WST-1 solution were added and cells were incubated for an additional 3 h at 37°C. Finally, the absorbance was read at 450 nm with a microplate ELISA (enzyme-linked immunosorbent assay) reader (ELx800, BioTek, USA) and the results were analyzed using the Gen5 software (BioTek, Winooski,Vermont). The reaction is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. The quantity of formazan dye in the medium is directly proportional to the number of viable metabolically active cells. The percentage of cell viability was calculated by assuming that control (untreated) samples are 100% viable.
3
Monocyte and Neutrophil Migration Assay
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Human PBMCs were treated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h prior to stimulation with either LL-37 (5 µM) or sLL-37 (5 µM). TC supernatants were collected after 24 h and used in the bottom chamber of Transwell plates (Costar, Corning, NY, USA), to monitor the migration of either monocytic cells or neutrophils. Human THP-1 monocytes (3 × 105 cells/well) or human neutrophils (6 × 105 cells/well), in 200 µl RPMI 1640 medium supplemented with 10% FBS, were added to the upper chamber of the inserts of Transwell plates. An insert pore size of 5 µm was used for THP-1 cells, and 3 µm for human neutrophils as previously described (25 (link), 26 (link)), and incubated at 37°C in a humidified chamber with 5% of CO2 for 30 min. TC supernatants (600 µl) collected from stimulated human PBMC (described above) were added to the bottom chamber of the Transwell plates, and further incubated for an additional 2 h. The number of cells that migrated to the bottom chamber was counted using a Scepter™ 2.0 Handheld Automated Cell Counter, Millipore Ltd. (ON, Canada). Human recombinant chemokines MCP-1, IL-8, and GRO-α (30 ng/ml each) were used in the bottom chamber in the migration assays as positive controls.
4
Isolation and Preservation of Lymphocytes
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Peripheral blood from the enrolled subjects was sampled into heparin-containing tubes. Mononuclear cells were immediately isolated using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Sweden), and were then suspended in medium containing RPMI-1640 (Life Technologies; USA), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (Life Technologies, USA). We isolated lymphocytes by negative selection using CD14-positive selection system (MACS system, Miltneyi Biotec Inc.) if PBMCs was more than 5 x 106 cell/mm3. Otherwise, we used PBMCs for further experiments for avoiding cells loss during lymphocyte selection. The intracellular cytokine responses in CD14-negative lymphocytes were similar to those in PBMCs, which were lower than those in peripheral blood leukocytes (Additional file 1 : Figure S1, supplement file). All cells were immediately frozen using a CELL-BANKER system (ZENOAQ, Japan) following the manufacturer’s instructions. The cells were then stored at -80 °C and defrosted within days of the scheduled experiments. Before the experiments, viable cells were counted using a Scepter™ 2.0 Handheld Automated Cell Counter (Millipore Corporation, Billerica, MA, USA).
5
Colony Formation Assay Protocol
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Cells were transfected with corresponding oligonucleotides or plasmids, and cultured at 37 °C for 24 h. Then, cells were digested with 0.25% trypsin and made into single-cell suspensions with DMEM. Cell number was counted with Scepter 2.0 Handheld Automated Cell Counter (Millipore). In all, 500 cells per well were seeded in a six-well plate. After about 2 weeks of culture, the colonies were fixed using methanol, dyed with 5% crystal violet and counted.
6
Cell Proliferation under Hypoxia and Epigenetic Modulators
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A549 cells were seeded in 6-well plates at the density of 6 × 103 cells mL−1. Once cells were attached, they were treated with 1 µM TSA, 20 mM NAM separately and in combination under both normoxia and hypoxia for 24 h and 48 h. Untreated cells cultured under both normoxia and hypoxia were used as controls. After the incubations, cells were washed with PBS, trypsinized, centrifuged and resuspended in PBS. Cell proliferation was determined by direct cell counting with Scepter™ 2.0 Handheld Automated Cell Counter (Millipore, Burlington, MA, USA).
7
Tau 244-378 Fibrils Metabolic Impact
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The metabolic influence of tau 244-378 amyloid fibrils was evaluated with MTT assay. 10.000/well ScN2a cells were plated in a 96-well, and the day after, different concentrations of sonicated tau 244-378 fibrils were directly added to the medium, and cells were incubated for 72 h. Cells were then incubated for 3 h at 37 °C with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) solution in PBS 1× followed by the solubilization with 1:1 DMSO/2-Propanol solution. The absorbance was measured at 570 nm in Enspire multimode plate reader (PerkinElmer) with a reference wavelength of 650 nm. Each condition was tested in six replicates and in three independent experiments.
Tau 244-378 fibril effects on proliferation and cell death were assessed by cell counting. 15.000/well ScN2a RML cells were plated in a 12-well, and the day after, they were treated with 2 μM tau 244-378 fibrils. Cells were left in incubation up to 72 h, after which they were detached with trypsin and counted using Scepter 2.0 Handheld Automated Cell Counter (Millipore) with 60 μM sensors. Three independent experiments were conducted, each one in three technical replicates.
Tau 244-378 fibril effects on proliferation and cell death were assessed by cell counting. 15.000/well ScN2a RML cells were plated in a 12-well, and the day after, they were treated with 2 μM tau 244-378 fibrils. Cells were left in incubation up to 72 h, after which they were detached with trypsin and counted using Scepter 2.0 Handheld Automated Cell Counter (Millipore) with 60 μM sensors. Three independent experiments were conducted, each one in three technical replicates.
8
PBMC Isolation from Peripheral Blood
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We followed the PBMC isolation methods of Shu et al. [12 (link)]. Briefly, peripheral blood was sampled in a heparin-containing tube from enrolled participants. We used the Ficoll-Paque PLUS (GE Healthcare Life Sciences, Sweden) to isolate mononuclear cells. Mononuclear cells were then suspended in medium containing RPMI-1640 (Life Technologies; U.S.A.), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (Life Technologies, USA). We then counted the viable cell by Scepter™ 2.0 Handheld Automated Cell Counter (Millipore Corporation, Billerica, MA, USA).
9
Apoptosis Induction Assay in Cells
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Cells were trypsinized in the culture medium and cell density was measured immediately using a Scepter 2.0 Handheld Automated Cell Counter (Milipore, USA). Subsequently, cells were dispensed into 96-well culture plates (Costar, USA) using an automatic cell dispenser (Multidrop Combi, Thermo, USA), incubated for ∼12 h to recover and subsequently transfected with Lipofectamine 2000 (Invitrogen, USA). The plasmid or RNA concentration used for transfection was 0.15 μg per well of 96-well plates. The media was changed 4 h after transfection to normal fresh medium to remove the transfection reagent and the cells were subsequently incubated for 20 h (DMEM with 100 units/ml penicillin and 0.1 mg/ml streptomycin without FBS or other protein supplement provided for FBS starvation group) to induce apoptosis. As control, a fresh aliquot of the same medium with 10% FBS was used. After about 20 h of starvation, CCK8 reagent (Cell Counting Kit (CCK-8/WST-8), QC-Bio, China) was added and the wells were screened using an automatic microplate reader (Enspire, Multimode Reader, USA). The absorbance at 450 nm was recorded.
10
Isolation of Peripheral Blood Mononuclear Cells
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Fifty ml of whole blood were collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson & Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMC) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA) and washed twice in PBS at 1500 RPM for 10 min; viable leukocytes were determined using a Scepter 2.0 Handheld Automated Cell Counter (Millipore, Billerica, MA).
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