Countess 3 automated cell counter

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Countess 3 Automated Cell Counter is a laboratory instrument designed to automate the process of counting and analyzing cells. It utilizes advanced imaging technology to provide accurate and reproducible cell counts and viability measurements.

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56 protocols using countess 3 automated cell counter

Cell Proliferation Assay after Poly(I:C) Transfection

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Cells were plated in 24-well plates and co-transfected with plasmids with or without Poly(I:C) as described above. Cell proliferation was assessed 24 hours after transfection. In brief, cells were trypsinized and mixed with an equal volume of 0.4% trypan blue stain, then subjected to cell counting using a Countess 3 automated cell counter (Thermo Fisher Scientific). All experiments were conducted in triplicate.

Geng H., Subramanian S., Wu L., Bu H.F., Wang X., Du C., De Plaen I.G, & Tan X.D. (2021). SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells. Frontiers in Immunology, 12, 679482.

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Flow Cytometry Analysis of Apoptosis

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MV4;11 cells were counted on a Countess 3 Automated Cell Counter (Thermo Fisher, UK) with the addition of trypan blue. Cells (1x10⁶) were aliquoted, spun down and resuspended in RPMI media containing test compounds at indicated concentrations. Additionally, QVD OPh (Sigma-Aldrich, UK) and Necrostatin-1 (Sigma-Aldrich) were added to the SIM1 treatment at 20μM final concentration. Treated cells were left to incubate for 24h at 37°C and 5% CO₂. On the following day, the cells were collected in a Falcon tube and spun down at 500g for 5 min. Supernatant was aspirated and cells were washed once in 1mL FACS buffer (PBS, 5% FBS, 0.05 % NaN) and afterwards resuspended in 100μL of the same buffer containing Apotracker Green (Biolegend, UK) and DAPI (Sigma-Aldrich) at final concentration of 400nM and 1μg/mL, respectively. Cells were incubated for 20 min on ice and afterwards washed in 1mL of FACS buffer and finally resuspended in 500μL of the same buffer. Measurements were done on BD FACS Canto II flow cytometer (Flow Cytometry and Cell sorting facility, University of Dundee, UK) using blue (ex: 488 nm; em: 530±30 nm) and violet (ex: 405 nm; em: 450±50 nm) laser for detection of FITC and DAPI, respectively. Data were analysed on FlowJo™ 10.7.1. Software and GraphPad Prism. Gating strategy is detailed in Supplementary Figure 1.

Imaide S., Riching K.M., Makukhin N., Vetma V., Whitworth C., Hughes S.J., Trainor N., Mahan S.D., Murphy N., Cowan A.D., Chan K.H., Craigon C., Testa A., Maniaci C., Urh M., Daniels D.L, & Ciulli A. (2021). Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity. Nature chemical biology, 17(11), 1157-1167.

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Proliferation Dynamics of Parental and Resistant Cells under Sotorasib Treatment

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To compare the proliferation rates between parental and resistant cells in the presence or absence of sotorasib, the cells were seeded at the density of 5 × 10⁴ cells/well in 6-well culture plates in triplicate for each condition. At the indicated time points, the cells were trypsinized, concentrated through centrifugation at 1000 rpm for 5 min, and resuspended in 20 μL of phosphate-buffered saline (PBS). The cells were stained with 20 μL of trypan blue solution [0.4% (w/v); Sigma-Aldrich, St. Louis, MO, USA] and were counted using an Invitrogen Countess 3 Automated Cell Counter (Thermo Fisher Scientific). To determine whether the inhibitors could rescue or aggravate the death of resistant cells upon sotorasib withdrawal, the cells were seeded at density of 2 × 10³ cells/well in 12-well culture plates in triplicate for each condition and were treated with different doses of the inhibitors for the indicated days, and cell numbers were counted as per the method described in the earlier text.

Chiou L.W., Chan C.H., Jhuang Y.L., Yang C.Y, & Jeng Y.M. (2023). DNA replication stress and mitotic catastrophe mediate sotorasib addiction in KRASG12C-mutant cancer. Journal of Biomedical Science, 30, 50.

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Culturing B16F10 and HEM cells

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The B16F10 mouse melanoma cell line and human epidermal melanocytes (HEM) were used. B16F10 cells were obtained from RIKEN (Tsukuba, Japan) and cultured in Roswell Park Memorial Institute (RPMI 1640) growth medium (Gibco, San Francisco, CA, USA) supplemented with fetal bovine serum (FBS, Gibco, USA) and 0.1% penicillin/streptomycin (Gibco, San Francisco, CA, USA).
HEM cells were purchased from Cell Applications and cultured in melanocyte growth medium: all-in-one ready-to-use (Cell Applications, Inc., San Diego, CA, USA). The cells were isolated from normal, healthy human neonatal foreskin.
The medium was changed every other day, and it was passaged when it reached 80–90% confluency using TrypLE Express (Gibco, San Francisco, CA, USA) and a Subculture Reagent Kit (HBSS, Trypsin/EDTA, and Trypsin Neutralizing Solution, Cell Applications, Inc., San Diego, CA, USA) each. The cells were kept under sterile conditions at 37 °C in a 75 cm² flask (BD Falcon, Becton Dickinson, Oxford, UK) in a humidified atmosphere of 5% CO₂. The cell viability was determined by trypan blue exclusion using a Countess 3 automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA).

Cho J., Bejaoui M., Tominaga K, & Isoda H. (2024). Comparative Analysis of Olive-Derived Phenolic Compounds’ Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes. International Journal of Molecular Sciences, 25(8), 4479.

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Inhibition of CaMKII Activity in NALM6 Cells

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NALM6 cells were grown in 6-well dishes and treated with varying concentration of water-soluble KN-93 (Calbiochem, Fisher Scientific). The range of KN-93 used in this study is identical to that which was previously shown to competitively inhibit both endogenous mammalian and zebrafish CaMKII activity and cell behaviors [15 (link),17 (link),67 (link),68 (link),88 (link)]. Cell number was assessed at 24-hour intervals until 72hpf using the Countess 3 Automated Cell Counter (Thermo Fisher).

Rothschild S.C., Lai G., Tombes R.M, & Clements W.K. (2023). Constitutively active CaMKII Drives B lineage acute lymphoblastic leukemia/lymphoma in tp53 mutant zebrafish. PLOS Genetics, 19(12), e1011102.

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Doxycycline-Induced Cell Proliferation

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MCF10A PIK3CA^H1047R, MCF7 and T47D cells stably expressing Tet-FRNK-EGFP, or pIND20 AFMID-202 were seeded evenly in a 48-well plate (10⁴), in 3 biological replicates for each condition, 24 hours prior to the initiation assay. For the cell lines stably bearing doxycycline-induced vectors, full or 1 mg/mL doxycycline-containing media was added. The cell number was counted at day 0, 3, and 6with a Countess 3 automated cell counter (Thermofisher Scientific, USA).

Ladewig E., Michelini F., Jhaveri K., Castel P., Carmona J., Fairchild L., Zuniga A.G., Arruabarrena-Aristorena A., Cocco E., Blawski R., Kittane S., Zhang Y., Sallaku M., Baldino L., Hristidis V., Chandarlapaty S., Abdel-Wahab O., Leslie C., Scaltriti M, & Toska E. (2022). The Oncogenic PI3K-Induced Transcriptomic Landscape Reveals Key Functions in Splicing and Gene Expression Regulation. Cancer Research, 82(12), 2269-2280.

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PBMC Isolation from Fresh Blood

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Fresh blood was used for the extraction of PBMC to ensure high viability of the cell sample. Ficoll–Paque density gradient media (15 mL) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was added to a 50 mL conical tube. In a separate 15 mL conical tube, 7–10 mL of fresh whole blood was mixed with Dulbecco’s Phosphate Buffered Saline (DPBS) in a 1:1 ratio and inverted four times to mix. The blood:DPBS mixture was then slowly layered above the Ficoll–Paque density gradient media to prevent mixing. Once layered, the 50 mL conical tube containing fresh blood and Ficoll–Paque density gradient media was spun down at 400× g for 40 min with the centrifuge brake off. Once spun, the plasma layer of the solution was removed, and the buffy coat was extracted and placed in a conical tube. The removed buffy coat was then mixed with DPBS at 3× volume of the buffy coat (1 mL buffy coat: 3 mL DPBS) to wash cells. Cells were then spun down at 400× g for five minutes. The wash step was repeated three times. Then, cells were counted using a Countess 3 Automated Cell Counter (ThermoFisher Scientific; Waltham, MA, USA) and cells were then resuspended at a concentration of 1.0 × 10⁶ cells/mL. PBMC were removed and immediately used for phenotyping.

Kisiolek J.N., Flores V.A., Ramani A., Butler B., Haughian J.M, & Stewart L.K. (2023). Eight Weeks of Daily Cannabidiol Supplementation Improves Sleep Quality and Immune Cell Cytotoxicity. Nutrients, 15(19), 4173.

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Glucose and Lactate Metabolism Assay

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Cells were seeded into 6-well plates and the DMEM medium was changed every 12 h. 500 μl of the medium was collected at 0 and 8 h of incubation to measure the initial and final concentrations of glucose and lactate, respectively, using a Silman M900 bioprocess biochemistry analyzer. Finally, the cells were digested with 0.25% trypsin solution (Thermo Fisher Scientific, USA) and counted through the countess 3 automated cell counter (Thermo Fisher Scientific, USA). The lactate and glucose levels were normalized based on cell count.

Song Q., Zhang K., Sun T., Xu C., Zhao W, & Zhang Z. (2023). Knockout of ENO1 leads to metabolism reprogramming and tumor retardation in pancreatic cancer. Frontiers in Oncology, 13, 1119886.

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Hematological Analysis of Treated Rats

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Blood was obtained in a glass vial containing anticoagulant (EDTA) on the tenth day following the animals’ sacrifice in order to analyze the hematological parameters. A standard technique was used to determine the total and differential counts of white blood cells (WBC) in normal, infected, and treated groups of rats with Countess 3 Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA) [48 ].

Asghar Z., Jamshaid T., Jamshaid U., Madni A., Akhtar N., Lashkar M.O, & Gad H.A. (2024). In Vivo Evaluation of Miconazole-Nitrate-Loaded Transethosomal Gel Using a Rat Model Infected with Candida albicans. Pharmaceuticals, 17(5), 546.

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Tet-Induced Proliferation Kinetics

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MCF10A PIK3CA^H1047R, MCF7, and T47D cells stably expressing Tet-FRNK-EGFP, or pIND20 AFMID-202 were seeded evenly in a 48-well plate (10⁴), in 3 biological replicates for each condition, 24 hours prior to the initiation assay. For the cell lines stably bearing doxycycline-induced vectors, full or 1 mg/mL doxycycline-containing media was added. The cell number was counted at day 0, 3, and 6 with a Countess 3 Automated Cell Counter (Thermofisher Scientific).

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