Cellometer auto 2000

Manufactured by Revvity

Sourced in United States, Germany

The Cellometer Auto 2000 is a cell counting and analysis instrument designed for automated cell counting and viability analysis. It utilizes image-based detection technology to provide accurate and reproducible cell counts across a wide range of cell types and sample volumes.

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Lab products found in correlation

Gentlemacs dissociator, by Miltenyi Biotec (8 mentions) Chromium controller, by 10x Genomics (6 mentions) Trypsin edta, by Thermo Fisher Scientific (6 mentions) Collagenase a, by Merck Group (5 mentions) Viastain aopi staining solution, by Revvity (4 mentions) Dead cell removal kit, by Miltenyi Biotec (4 mentions) Qubit 2.0 dna hs assay, by Thermo Fisher Scientific (3 mentions) Ack lysis solution, by Thermo Fisher Scientific (3 mentions) Ionomycin, by Merck Group (3 mentions) Aopi live dead dye, by Revvity (3 mentions) Celltrace violet ctv proliferation dye, by Thermo Fisher Scientific (2 mentions) Soluble rhil 2, by ProSpec (2 mentions) Soluble il 2, by ProSpec (2 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions) Lsrfortessa, by BD (2 mentions) Totalseq, by BioLegend (2 mentions) Tapestation high sensitivity d1000 assay, by Agilent Technologies (2 mentions) Minielute reaction cleanup kit, by Qiagen (2 mentions) Quantstudio 5 system, by Thermo Fisher Scientific (2 mentions) Cell strainer, by Miltenyi Biotec (2 mentions)

102 protocols using cellometer auto 2000

Amniotic Fluid Leukocyte Isolation and Characterization

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This was a cross-sectional study of women who underwent transabdominal amniocentesis due to clinical indications or sampling of amniotic fluid during cesarean section. Women were enrolled at Hutzel Women’s Hospital of the Detroit Medical Center. Amniotic fluid samples were acquired by an automatic cell counter (Cellometer Auto 2000, Nexcelom Bioscience, Lawrence, MA, USA) to obtain the viable cell numbers, most of which are white blood cells or leukocytes [48 (link)]. The inclusion criteria were as follows: 1) amniotic fluid samples without blood contamination and 2) amniotic fluid samples with a large number of viable leukocytes (including mostly monocytes and neutrophils [48 (link)]) (>1 × 10⁵ cells/mL) to perform fluorescence-activated cell sorting (FACS) of amniotic fluid monocytes/macrophages.
All of the women provided written informed consent to donate additional amniotic fluid for research purposes, according to protocols approved by the Institutional Review Boards of the Detroit Medical Center (Detroit, MI, USA), Wayne State University and the Perinatology Research Branch, an intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U. S. Department of Health and Human Services (NICHD/NIH/DHHS).

Gomez-Lopez N., Romero R., Leng Y., Xu Y., Slutsky R., Levenson D., Pacora P., Jung E., Panaitescu B, & Hsu C.D. (2019). The Origin of Amniotic Fluid Monocytes/Macrophages in Women with Intra-Amniotic Inflammation and/or Infection. Journal of perinatal medicine, 47(8), 822-840.

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Investigating MEK Inhibition in LPS-Induced Lung Inflammation

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C57Bl/6 mice were anesthesitized using isoflurane and positioned for
oropharyngeal E. coli LPS instillation (1.5 μg/g in 50 μl
sterile PBS). Mice were monitored daily for weight change and activity. Groups of mice
received MEKi PD0325901 (IP; 20 μg/kg in 10% DMSO) or vehicle control (IP; 10%
DMSO) on days 1 and 3 after LPS delivery. Mice were sacrificed at days 2 and 4 after LPS
challenge and subjected to three serial bronchoalveolar lavages with 0.9 ml PBS + 5 mM
EDTA. Total BAL cells were enumerated on a Cellometer Auto 2000 (Nexcelom Bioscience LLC,
Lawrence, MA) and staining by Diff-Quick was performed on cytospin preparations. BAL
macrophages were isolated by adherence to tissue culture plates for 1 hour with
non-adherent cells removed by washing with PBS. BAL macrophage RNA was isolated and used
as the template for cDNA that was then used in qPCR to determine relative gene expression.
Surface CD71 levels were measured by multicolor flow cytometry on BAL macrophages that
were identified as CD45⁺, Ly6G⁻, CD11c⁺,
SigF⁺ cells.

Long M.E., Eddy W.E., Gong K.Q., Lovelace-Macon L.L., McMahan R.S., Charron J., Liles W.C, & Manicone A.M. (2016). MEK1/2 Inhibition Promotes Macrophage Reparative Properties. Journal of immunology (Baltimore, Md. : 1950), 198(2), 862-872.

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Cell Isolation and Multiplexing Protocol

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Cells isolated from Peyer’s patches were counted using the Nexcelom Cellometer Auto 2000. An aliquot of 1,000,000 cells from each sample was centrifuged at 400 RCF for 10 minutes (4°C). The supernatant was discarded and the pellets were resuspended in 100ul of 10x Genomics Cell Multiplexing Oligo (CMO) and incubated at RT for 5 minutes. Stained cells were washed three times in 1mL of cold wash buffer (PBS + 1% BSA) by 4°C centrifugation at 400 RCF to remove unbound CMOs. Washed cells were resuspended in 200ul resuspension buffer (PBS + 0.05% BSA) and counted. Stained samples were pooled in equal cell numbers and centrifuged at 400 RCF for 5 minutes (4°C). Supernatant was removed and pellet was resuspended in 100ul of resuspension buffer.

Canales-Herrerias P., Uzzan M., Seki A., Czepielewski R.S., Verstockt B., Livanos A., Raso F., Dunn A., Dai D., Wang A., Al-taie Z., Martin J., Ko H.M., Tokuyama M., Tankelevich M., Meringer H., Cossarini F., Jha D., Krek A., Paulsen J.D., Nakadar M.Z., Wong J., Erlich E.C., Onufer E.J., Helmink B.A., Sharma K., Rosenstein A., Chung G., Dawson T., Juarez J., Yajnik V., Cerutti A., Faith J., Suarez-Farinas M., Argmann C., Petralia F., Randolph G.J., Polydorides A.D., Reboldi A., Colombel J.F, & Mehandru S. (2023). Gut-associated lymphoid tissue attrition associates with response to anti-α4β7 therapy in ulcerative colitis. bioRxiv.

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Cell Viability Assay with BCar

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Cells were plated at approximately 25% confluency in 6-well plates, treated with BCar at the indicated concentrations for 0–3 days, and then harvested by trypsinization. Equal volumes of the cell suspension and 0.4% solution of trypan blue in PBS were mixed to stain dead cells. Cell viability was determined using a Cellometer Auto 2000 (Nexcelom, Lawrence, MA) as instructed by the manufacturer.

Graff B.T., Palanivel C., Jenkins C.B., Baranowska-Kortylewicz J, & Yan Y. (2023). Benzimidazole carbamate induces cytotoxicity in breast cancer cells via two distinct cell death mechanisms. Cell Death Discovery, 9, 162.

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Cell Proliferation and Viability Assay

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Eight hundred thousand cells seeded into 6-well plates for 24 hours. Cells were treated with PQ1 at various concentrations or overexpressed with Cx43. After another 24, 48 or 72 hours tissue culture media of respective treatments was saved and 0.5 mL of trypsin was added to the cells for 5 minutes. Three mL of PBS was used to harvest cells. Cells were spun down for 5 minutes at 13,000 rpm; afterwards solution of tissue culture media, trypsin and PBS was removed. Nine hundred μL of PBS and 100 μL of trypan blue were added to the pellet and left to stand for 5 minutes. Cellometer Auto 2000 from Nexcelom Bioscience was used to measure number of cells for proliferation and viability.

Bigelow K, & Nguyen T.A. (2014). Increase of gap junction activities in SW480 human colorectal cancer cells. BMC Cancer, 14, 502.

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Isolation and Culturing of Mouse Embryonic Fibroblasts

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Pole4^+/-Trp53^+/- male and female mice in C57BL/6 background were mated. Pregnant females at 13.5 days gestation were subjected to euthanasia under anaesthesia, followed by uterine dissection to isolate individual embryos. Each embryo was washed in PBS followed by removal of head (used for genotyping) and internal organs (heart and liver). The embryo body was minced with sterile razor blades and incubated in trypsin at 37°C for 20 min, followed by gentle pipetting of the trypsin digest. Cell suspension was pelleted, resuspended and plated in 10 cm dishes (now considered passage 0) in DMEM (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% FBS (SIGMA) and 50μg/mL penicillin-streptomycin, 2mM L-glutamine. Once subconfluent, a standard 3T3 protocol was followed: every 3 days cells were trypsinized, counted using cellometer Auto 2000 (Nexcelom Bioscience) to determine the number of Population doublings (PD) and then replated at a fixed density (8x10⁵ cells per 100-mm dish). The accumulation of population doubling level (PDL) was calculated using the formula ΔPDL = log(nh/ni)/log2, where ni is the initial number of cells and nh is the cell number at each passage.

Borel V., Boeing S., Van Wietmarschen N., Sridharan S., Hill B.R., Ombrato L., Perez-Lloret J., Jackson D., Goldstone R., Boulton S.J., Nussenzweig A, & Bellelli R. (2022). Disrupted control of origin activation compromises genome integrity upon destabilization of Polε and dysfunction of the TRP53-CDKN1A/P21 axis. Cell Reports, 39(9), 110871.

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Isolation and Cryopreservation of PBMCs and CD45+ Cells from Aortic Tissue

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PBMCs were separated by standard Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation, and then cryopreserved in FBS with 10% DMSO (Sigma-Aldrich, St. Louis, MO, USA) until use. To purify single-cell suspensions of lymphocytes from tissue samples, aortic tissue was placed in a gentle MACS C-Tube (Miltenyi Biotec, Bergisch Gladbach, Germany) enzyme H, enzyme R, and enzyme A (Miltenyi Biotec) pre-mixed in RPMI. These C-tubes were then placed on a gentle MACS Octo Dissociator. Next, the cell suspension was passed through a 70-μm cell strainer, and the cells were washed once and then resuspended in media. The number and viability of mononuclear cells assessed using a Cellometer^® Auto 2000 (Nexcelom, Lawrence, MA, USA). The cells were then washed once and cryopreserved. CD45⁺ cells from aortic tissue were purified from single-cell isolates using the REAlease CD45 (TIL) MicroBead Kit (130-121-563; Miltenyi Biotec).

Seo I.H., Lee S.J., Noh T.W., Kim J.H., Joo H.C., Shin E.C., Park S.H, & Ko Y.G. (2021). Increase of Vδ2+ T Cells That Robustly Produce IL-17A in Advanced Abdominal Aortic Aneurysm Tissues. Immune Network, 21(2), e17.

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Thymocyte Isolation and Enumeration

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Harvested thymi were crushed and filtered through a 40 μM cell strainer. Cells were counted using Cellometer^® Auto 2000 (Nexcelom Bioscience) at 1:1 dilution in ViaStain^TM AOPI staining solution (Nexcelom Bioscience). Red blood cells were lysed using red blood cell lysis buffer. Cells were fixed, stained, and analyzed as described for the bone marrow.

Han H., Yan H, & King K.Y. (2021). Broad-Spectrum Antibiotics Deplete Bone Marrow Regulatory T Cells. Cells, 10(2), 277.

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Single-cell ATAC-seq profiling of CD8+ T cells

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CD8⁺ T cells were isolated from the islets and ndLN of 4 E8i^CRE-GFP.NOD (8-week females). Samples were viability stained in PBS and surface stained for sorting in sort buffer (contains PBS, EDTA, BSA and HEPEs) with 10% normal mouse serum. Cells were sorted on Lymphocytes, single cell, Thy1.2⁺, CD8⁺ T cells. Cells were sorted into 1.5 mL Eppendorf tubes containing cRPMI and nuclei were isolated in accordance with the Nuclei Isolation for Single Cell ATAC Sequencing 10x Genomics demonstrated protocol, using the adaptations for low cell number input. Nuclei suspended in 0.04% bovine serum albumen (BSA, Sigma), counted using Cellometer Auto2000 (Nexcelom), and loaded into Single Cell Chip and processed through 10X controller for droplet generation and Library preparation.

Grebinoski S., Zhang Q., Cillo A.R., Manne S., Xiao H., Brunazzi E.A., Tabib T., Cardello C., Lian C.G., Murphy G.F., Lafyatis R., Wherry E.J., Das J., Workman C.J, & Vignali D.A. (2022). Autoreactive CD8+ T cells are restrained by an exhaustion-like program that is maintained by LAG3. Nature immunology, 23(6), 868-877.

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Immune Profiling of the Tumor Microenvironment

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For ELISpot and intracellular cytokine staining (ICS) by flow cytometry, spleens were collected and crushed through a 70μM cell strainer to form a single cell suspension. Red blood cells were lysed (Tonbo Biosciences, San Diego, CA) and splenocytes were counted (Cellometer Auto 2000, Nexcelom Bioscience, Lawrence MA). To analyze the TME, mice were treated with brefeldin A, as previously described (21 (link)) (Sigma-Aldrich, St. Louis, MO, 250μg per mouse) for four to six hours before euthanization. Tumors were collected, weighed, processed (Tumor Dissociation Kit and gentleMACS dissociator, Miltenyi Biotec, Germany), and total cells were counted.

Buchta Rosean C., Leyder E.C., Hamilton J., Carter J.J., Galloway D.A., Koelle D.M., Nghiem P, & Heiland T. (2023). LAMP1 targeting of the large T antigen of Merkel cell polyomavirus results in potent CD4 T cell responses and tumor inhibition. Frontiers in Immunology, 14, 1253568.

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