Nucleocounter nc 100

Manufactured by ChemoMetec

Sourced in Denmark

The NucleoCounter NC-100 is a cell counter that uses fluorescence microscopy to determine the total cell count and viability of a cell sample. It provides fast and accurate cell counting results.

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NucleoCounter (85 citations) NucleoCounter® NC-100 TM system (4 citations) NucleoCounter® NC-100TM (2 citations) NC-100™ NucleoCounter® Automated Cell Counting System (2 citations) NucleoCounter NC-100 automated cell count system (2 citations)

61 protocols using nucleocounter nc 100

Characterization of Cell Line Panel

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Murine melanoma B16-F10 (CRL-6475), murine colon CT26 (CRL-2638), human melanoma A375 (CRL-1619), human lung A549 (CCL-185), human prostate DU145 (HTB-81), human prostate PC-3 (CRL-1435) and human breast MCF7 (HTB-22) were purchased from ATCC. B16-F10 or A375 were cultured in D MEM (Gibco, #10569010). CT26 was cultured in RPMI-1640 (Gibco, # 72400047). A549 and PC-3 cells were cultured in Ham's F-12K (Gibco, #21127022). DU145 and MCF7 were cultured in MEM (Gibco, # 42360099). All culture media are supplemented with 10% FBS (HyClone, #SH30071.03). Human recombinant insulin (10 µg ml⁻¹, Gibco #12585014) was additionally included in the culture media for MCF7. All cells were maintained at 37 °C, 5% CO₂, and 95% relative humidity. The cells were routinely tested for mycoplasma. The CRISPR-edited B16-F10 and CT26 cell lines were PCR-evaluated by IDEXX BioAnalytics to be free of viral contamination. The CRISPR-edited B16-F10 lines were genetically confirmed as mouse origin, and had almost identical short tandem repeat profile (> 90% match) to that established for B16-F10 (ATCC, CRL-6475). Cell number was determined using NC-100 NucleoCounter (ChemoMetec).

Ng S., Lim S., Sim A.C., Mangadu R., Lau A., Zhang C., Martinez S.B., Chandramohan A., Lim U.M., Ho S.S., Chang S.C., Gopal P., Hong L.Z., Schwaid A., Fernandis A.Z., Loboda A., Li C., Phan U., Henry B, & Partridge A.W. (2022). STUB1 is an intracellular checkpoint for interferon gamma sensing. Scientific Reports, 12, 14087.

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Passaging and Expansion of Mammalian Cells

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A WCB vial of single cells was thawed from liquid Nitrogen storage and used to seed a monolayer triple flask (500 cm², Thermo Scientific Loughborough, Leicestershire) in antibiotic-free culture media (MEMalpha, PAA, Pasching, Austria) supplemented with Foetal Calf Serum (10%, PAA, Pasching, Austria) and insulin (0.27 IU/mL, Novo Nordisk, Bagsværd, Denmark) and passaged after 4 to 7 days growth. Cells were acclimatised to supplemented antibiotics during the second passage (penicillin/streptomycin (45 u/ml pen, 45 µg/ml strep), Thermo Fisher Scientific: Life Technologies, Paisley, Scotland) and fungizone (1.1 µg/mL, Thermo Fisher Scientific:Life Technologies, Paisley, Scotland), and at the third passage used to seed 7 x Cell Factories (Easy Fill 10, 6320 cm², Thermo Scientific, Loughborough, Leicestershire). Cells (139 × 10⁶ cells in 1.7 L culture media) were used per Cell Factory and grown for 6 days, and then harvested using TrypLE Select (Thermo Fisher Scientific:Life Technologies, Paisley, Scotland) dissociation reagent and pelleted at 289rcf (1150 rpm, Thermo Scientific Multifuge X3-R; 4-head swing out rotor) (500 mL Corning centrifuge tubes). Resuspended cells were counted for viable cell number using an NC-100 Nucleocounter (Chemometec, DK-3450 Allerod, Denmark), and then mixed in suspension with alginate for encapsulation.

Selden C., Bundy J., Erro E., Puschmann E., Miller M., Kahn D., Hodgson H., Fuller B., Gonzalez-Molina J., Le Lay A., Gibbons S., Chalmers S., Modi S., Thomas A., Kilbride P., Isaacs A., Ginsburg R., Ilsley H., Thomson D., Chinnery G., Mankahla N., Loo L, & Spearman C.W. (2017). A clinical-scale BioArtificial Liver, developed for GMP, improved clinical parameters of liver function in porcine liver failure. Scientific Reports, 7, 14518.

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Murine NPC-derived Astrocyte Isolation

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Murine NPC-derived astrocytes were plated in 10-cm² plates
coated with human fibronectin (2.5 ug/mL; Millipore) at a density of
6×10^6 cells per dish. Media was changed every 2 days. After 4
days, cells were washed 2× with PBS and incubated with Accutase (Gibco)
to lift cells from the culture dish. Accutase was neutralized with culture
media, and cells were centrifuged for 5 minutes at 500 RCF. The resulting cell
pellets were re-suspended in 1 mL of 1× PBS and viability was determined
using an NC-100 Nucleo-counter (Chemometec), according to the
manufacturer’s protocol.

Heilman P.L., Song S., Miranda C.J., Meyer K., Srivastava A.K., Knapp A., Wier C.G., Kaspar B.K, & Kolb S.J. (2017). HSPB1 mutations causing hereditary neuropathy in humans disrupt non-cell autonomous protection of motor neurons. Experimental neurology, 297, 101-109.

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Expansion and Proliferation of PSC

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PSC were reduced to single cell suspension by 5 min incubation with accutase (L0950; Biowest, Nuaillé, France), the number of viable cells determined by an automated cell counter (NC-100 NucleoCounter; Chemometec, Allerod, Denmark) and seeded at 15,000 cells/cm² in StemMACS iPS-Brew XF supplemented with 10 µM Y-27,632 Rock inhibitor (72,302; STEMCELL Technologies, Vancouver, Canada). To calculate population doubling time, single cell suspensions were seeded in 96-well E-Plates (ACEA Biosciences, San Diego, CA, USA) at 5000 cells/cm² in StemMACS iPS-Brew XF supplemented with 10 µM Y-27,632 Rock inhibitor. Proliferation rate was determined based on the proprietary cell index parameter on xCELLigence Real Time Cell Analysis single plate system (RTCA-SP; ACEA Biosciences; RRID: SCR_014821) as already described [52] .

Barilani M., Cherubini A., Peli V., Polveraccio F., Bollati V., Guffanti F., Del Gobbo A., Lavazza C., Giovanelli S., Elvassore N, & Lazzari L. (2020). A circular RNA map for human induced pluripotent stem cells of foetal origin. EBioMedicine, 57, 102848.

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PBMC Isolation and Cryopreservation

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Blood was drawn into CPT tubes (BD Biosciences, Oxford, UK) and spun at site to isolate PBMCs. The PBMCs were shipped to the biomarker repository at the Cancer Research UK Liverpool Clinical Trials Unit-GCP Laboratory Facility, UK. Viability and cell count of the PBMCs was measured using a ChemoMetec (Allerod, Denmark) NucleoCounter NC-100 before freezing in 90% DMSO and 10% human serum to −80 °C overnight. The aliquots were then stored at −150 °C for subsequent batch analysis. Blood for serum was collected into SST tubes (BD Biosciences) centrifuged at 1500 g for 10 min, aliquoted and stored at −80 °C.

Middleton G., Greenhalf W., Costello E., Shaw V., Cox T., Ghaneh P., Palmer D.H, & Neoptolemos J.P. (2016). Immunobiological effects of gemcitabine and capecitabine combination chemotherapy in advanced pancreatic ductal adenocarcinoma. British Journal of Cancer, 114(5), 510-518.

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Measuring NIS-Mediated Radionuclide Uptake

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cNIS⁻ and cNIS⁺ myoblasts were plated in quadruplicate in a 24-well plate for growth or differentiation. On the day of analysis, cells were incubated with ^{99 m}TcO₄⁻ (185 kBq) in 200 μL DMEM for 1 h, specific NIS inhibition was obtained by addition of 10 μM sodium perchlorate (NaClO₄) in some wells. Medium was collected, along with the PBS used to rinse the cells. Cells were collected, counted with a NucleoCounter NC-100 (ChemoMetec), and lysed. Radioactivity of the cells and of the media was measured by a 2480 Wizard automatic gamma counter (PerkinElmer). Uptake values were corrected for each sample according to the number of cells. Data were presented as mean ± SEM. One-way ANOVA was performed for in vitro radiotracer uptake experiment. p values < 0.05 were considered statistically significant.

Punzón I., Mauduit D., Holvoet B., Thibaud J.L., de Fornel P., Deroose C.M., Blanchard-Gutton N., Vilquin J.T., Sampaolesi M., Barthélémy I, & Blot S. (2020). In Vivo Myoblasts Tracking Using the Sodium Iodide Symporter Gene Expression in Dogs. Molecular Therapy. Methods & Clinical Development, 17, 317-327.

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Isolation and Culture of Bone Marrow Mononuclear Cells

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50 ml bone marrow aspirate was obtained from the iliac crest by needle aspiration under local anesthesia. The sample was diluted 1:2 with phosphate-buffered saline pH 7.4 (PBS, −Ca²⁺ -Mg²⁺, Gibco, Invitrogen, Denmark, cat.no. 10010–015). Mononuclear cells (MNCs) were harvested by gradient centrifugation on Lymphoprep (1077 g/cm³, Medinor, Denmark, cat.no. 1114547), washed with PBS and counted using NucleoCounter® NC-100™ (Chemometec, Denmark) according to manufacturer’s instructions. Primary cell cultures of MNCs were established by seeding 2 × 10⁷ cells/T75-flask (Nunc, Thermo Scientific, Denmark, cat.no. 156494) in complete medium containing Dulbecco’s Modified Eagle Medium, low glucose (1 g/l) (DMEM) supplemented with 25 mM HEPES and L-Glutamin, (PAA Laboratories, Austria, cat.no. E15-808), 10% Fetal Bovine Serum Farma grade (FBS, PAA Laboratories, cat.no. A11-512) and 1% Penicillin/Streptomycin (Gibco, cat.no. 15140–122). The cells were incubated in standard conditions at 37°C in humid air with 5% CO₂. The medium was changed 5 days after initial seeding, and subsequently every 3–4 days.

Tratwal J., Follin B., Ekblond A., Kastrup J, & Haack-Sørensen M. (2014). Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF. BMC Molecular Biology, 15, 11.

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BPF Cytotoxicity on Osteosarcoma Cells

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Saos-2 and MG63 cells were seeded at a density 200,000 and 100,000 cells/well, respectively, in 6-well dishes. Cells were grown in serum-free medium, and incubated with BPF 0.001; 0.001; 0.1 mg/mL for 24 h. Then, cell number was determined using the Nucleo counter NC-100 (Chemometec A/S, Lillerød, Denmark).

Pujia A., Russo C., Maurotti S., Pujia R., Mollace V., Romeo S, & Montalcini T. (2018). Bergamot Polyphenol Fraction Exerts Effects on Bone Biology by Activating ERK 1/2 and Wnt/β-Catenin Pathway and Regulating Bone Biomarkers in Bone Cell Cultures. Nutrients, 10(9), 1305.

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Cell Counting and Viability Assay

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Cells were counted either by Trypan blue dye exclusion methods or with Perfect-Count Microspheres (Cytognos) in a FACSCalibur cytometer (Becton Dickinson). Viability was determined using the 7-amino-actinomycin D (7-AAD, BD Biosciences) exclusion method and expressed as a percentage (%) of total cells. Data were analyzed with the CellQuest Pro (Becton Dickinson) software. Alternatively, cell numbers and viability were determined using the NucleoCounter NC-100 device (ChemoMetec A/S, Denmark).

Grau-Vorster M., Laitinen A., Nystedt J, & Vives J. (2019). HLA-DR expression in clinical-grade bone marrow-derived multipotent mesenchymal stromal cells: a two-site study. Stem Cell Research & Therapy, 10, 164.

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DEPDC5 Knockdown Effect on LX-2 Cell Growth

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LX‐2 cells were seeded in six‐well plates. When 50% confluent, cells were transfected with DEPDC5 siRNA or scramble siRNA. Cell numbers were counted 24, 48, and 72 hours after transfection using the Nucleocounter NC‐100 (Chemometec A/S, Lillerød, Denmark).

Burza M.A., Motta B.M., Mancina R.M., Pingitore P., Pirazzi C., Lepore S.M., Spagnuolo R., Doldo P., Russo C., Lazzaro V., Fischer J., Berg T., Aghemo A., Cheroni C., De Francesco R., Fargion S., Colombo M., Datz C., Stickel F., Valenti L, & Romeo S. (2015). DEPDC5 variants increase fibrosis progression in Europeans with chronic hepatitis C virus infection. Hepatology (Baltimore, Md.), 63(2), 418-427.

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