20,000 cells were counted using Vi-CELL cell counter (Beckman Coulter) and seeded in 24-well plates and tetracycline (Bioshop TET 70125) was added to induce protein expression at final working concentration of 1 μg/mL. After 3 days, cells were lifted off using 100 μL trypsin and 37°C incubation for 3 minutes, after which 400 μL media was added to lift cells, which were then counted (Vi-CELL TM Cell counter, Beckman Coulter).
Vi cell cell counter
Manufactured by Beckman Coulter
Sourced in United States, Canada
The Vi-Cell cell counter is a compact, automated instrument designed for cell counting and viability analysis. It utilizes a sampling mechanism and image processing technology to determine the concentration and viability of cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Transwell permeable support, by Corning (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Auto hematology analyzer bc 5000, by Mindray (2 mentions)
Rpm1 1640, by Thermo Fisher Scientific (2 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions)
At7519, by Selleck Chemicals (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Thymidine, by Merck Group (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Muse annexin 5 dead cell assay kit, by Merck Group (2 mentions)
Muse cell analyzer, by Merck Group (2 mentions)
Cedex bioht, by Roche (2 mentions)
Atp bioluminescence assay kit cls 2, by Roche (2 mentions)
Amplex red glucose glucose oxidase assay kit, by Thermo Fisher Scientific (2 mentions)
7 aad, by BD (2 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (2 mentions)
Agencourt ampure xp, by Beckman Coulter (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Nextseq desktop sequencer, by Illumina (2 mentions)
72 protocols using vi cell cell counter
1
Cell Culture Induction and Analysis
2
Cell Viability and Growth Assay Protocol
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Unless stated otherwise, 3 × 103 cells were counted by a ViCell cell counter (Beckman Coulter, Brea, CA, USA), seeded in 96‐well plates, and treated with the respective compounds as indicated. For dose–response curves, serial dilutions of respective compounds were prepared in 100 µL medium and cells were added on top in 100 µL medium. Cell viability was assessed by the addition of Resazurin (Sigma, St. Louis, MO, USA) to final concentration of 50 µm , and fluorescence was measured 6 h later at 540 nm excitation/590 nm emission in a PerkinElmer Envision 2104 (PerkinElmer, Waltham, MA, USA) Multilabel plate reader. At least three wells per condition were averaged, and viability is presented as percentage relative to respective control. For growth analysis, 3 × 104 cells were seeded in 12‐well plates and counted by a ViCell cell counter (Beckman Coulter, Brea, CA, USA).
3
Cell Viability Assay Comparing Inhibitor Effects
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Information about all inhibitors used is listed in Table S3 . All inhibitors were prepared in DMSO as 10 or 20 mm stock. The effect of the various treatments on cell viability was evaluated by two methods. The first method used the Vi‐CELL cell counter (Beckman Coulter). Briefly, cells were plated in 24‐well plates at a density of 9.4 × 104 cells per well in triplicate. Cells were then treated with either vehicle (DMSO) or inhibitors. After 48 h, cells were trypsinized and cell viability was measured with a Vi‐CELL cell counter (Beckman Coulter). The percentage of viable cells was defined as the ratio of cell number in the inhibitor‐treated group to that of the DMSO‐treated group. IC50 was calculated using graphpad prism software (San Diego, CA, USA). We also used Cell Counting Kit‐8 (Dojindo) to quantify the cell viability according to the instruction manual.
4
Inducible ACO2 Protein Expression in HEK293 Cells
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20,000 HEK293 WT ACO2 cells were seeded in 24-well plates and Tetracycline (Bioshop TET 70125) was added to induce protein expression at final working concentration of 1 μg/mL. Pioglitazone hydrochloride (PZH; Sigma Aldrich E6910) was added at the indicated final concentration. After 3 days, cells were lifted off by using 100-μL trypsin and 37°C incubation for 3 minutes, after which 400-μL media was added to lift cells, which were counted (Vi-CELL TM Cell counter, Beckman Coulter).
5
Transwell-Based THP-1 Cell Migration Assay
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Migration of THP-1 cells was measured using Transwell Permeable Supports (Costar, Cambridge, MA, USA) as previously described [16 (link)]. Cells (5 × 105 cells in 100 μL of 0.1% BSA) were loaded into the top chamber of 5-μm-pore polycarbonate transwell inserts. Transwell chambers were inserted into wells filled with a supernatant isolated from THP-1 cells treated with 27OHChol with or without dexamethasone. After incubation for 3 h at 37°C, the number of cells that migrated to the bottom chamber was counted using a Vi-Cell cell counter (Beckman Coulter, Inc. Brea, CA, USA).
6
Isolation of Mouse Splenocytes
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Single-cell suspension of splenocytes were obtained by mechanical digestion through a 100 μm nylon cell strainer (Celltreat) into 5 ml of DMEM + 5% FBS. To remove erythrocytes and decrease the amount of apoptotic cells in cell preparations, 5 ml of splenocyte suspensions were then carefully added on top of 9 ml of Ficoll-PAQUE Premium (GE Healthcare), and centrifuged for at 2,300 RPM for 20 min at room temperature with no centrifuge brake. The leukocyte containing buffy coat layer was then collected and washed three times in cold DMEM + 5% FBS. Cell counts were performed using hemacytometer or automated Vi-CELL Cell Counter (Beckman Coulter).
7
Isolation and Activation of Human PBMCs
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Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood obtained from consenting healthy donors. Blood was collected in Sodium Heparin BD Vacutainer tubes and diluted 1:1 in PBS containing 2% FBS. The mixture was overlayed onto 15 mL Ficoll-Paque (GE Healthcare, Baie d'Urfe, QC, Canada) in SepMate tubes (Stemcell Technologies, Vancouver, BC, Canada) and spun at 1200g for 20 min. The buffy coat containing the PBMCs was collected and washed twice with PBS containing 2% FBS. Cells were counted on a Vi-CELL cell counter (Beckman Coulter, Mississauga, ON, Canada).
PBMCs were labeled with 1 μmol/L CFSE (Life Technologies) as per manufacturer's instructions and cultured in 96-well flat-bottom plates (105/well) coated with 5 μg/mL anti-CD3 mAb (OKT3, eBioscience, San Diego, CA, USA) in RPMI containing 10% FBS (Life Technologies) and 250 IU/mL recombinant human IL-2 (Proleukin, Chiron Corporation, Emeryville, CA, USA). TAT-G-Gpep, TAT-G-Gpep-Sc, or IFN-β (Bio Basic Inc., Markham, ON, Canada) were added to the wells and cultured for 3 days. Cells were subsequently harvested and stained for flow cytometry.
PBMCs were labeled with 1 μmol/L CFSE (Life Technologies) as per manufacturer's instructions and cultured in 96-well flat-bottom plates (105/well) coated with 5 μg/mL anti-CD3 mAb (OKT3, eBioscience, San Diego, CA, USA) in RPMI containing 10% FBS (Life Technologies) and 250 IU/mL recombinant human IL-2 (Proleukin, Chiron Corporation, Emeryville, CA, USA). TAT-G-Gpep, TAT-G-Gpep-Sc, or IFN-β (Bio Basic Inc., Markham, ON, Canada) were added to the wells and cultured for 3 days. Cells were subsequently harvested and stained for flow cytometry.
8
Hematological Analysis of Peripheral Blood and Bone Marrow
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Hematologic parameters in PB after tail bleeding were analyzed by Auto Hematology Analyzer BC-5000 (MINDRAY). BM cells were harvested from one femur and suspended in HBSS+ buffer on ice before evaluating by Vi-CELL Cell Counter (Beckman).
9
Trypan Blue Assay for Cell Viability
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The lytic capacity was measured using the trypan blue exclusion method. Human tumor cells were transduced in suspension by respective chimeric viruses at the indicated MOI. A total of 3 × 105 cells/well were plated in 6-well culture dishes in 2 mL of medium supplemented with 10% FCS. Cells were then cultured at 37 °C for 5 days and the viable cells were counted by trypan blue exclusion using a Vi-Cell Cell Counter (Beckman Coulter, Brea, CA, USA). All samples were analyzed in triplicate. Mock-infected cells served as negative control and established the 100% survival point for the given assay.
10
Magnetic Cell Separation Protocol
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Cells were harvested by trypsinization and recovered by centrifugation (200 g, 5 min). The cell pellet was resuspended in 1.4 mL RPMI 1640, transferred into a 1.5 mL Eppendorf tube, and put into a 16-Tube SureBeads™ Magnetic Rack (Bio-Rad, Munich, Germany), which was refrigerated overnight. The non-magnetic cell fraction (supernatant) was then collected and transferred into a fresh tube. Loosely attached or sedimented cells were recovered by washing the bottom of the tube in the magnetic rack twice with 15 µL DPBS and adding this to the original supernatant. Magnetic cells were recovered after removal of the tube from the magnetic rack by resuspension in 1.4 mL DPBS. Cell densities in both suspensions were determined using a Vi-Cell cell counter (Beckman Coulter, Krefeld, Germany).
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