Hybrid cell count module

Manufactured by Keyence

Sourced in Japan

The Hybrid Cell Count module is a compact and versatile device designed for cell counting and analysis. It utilizes a combination of optical and impedance-based technologies to accurately determine cell concentration and viability. The module provides precise measurements of cell count, cell size, and other key parameters, enabling efficient monitoring and analysis of cell cultures.

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Lab products found in correlation

Pannoramic flash 3 system, by 3DHISTECH (2 mentions) Bz x700 microscope, by Keyence (2 mentions) Bz x800 analyzer software, by Keyence (1 mentions) Bz x700 all in one microscope, by Keyence (1 mentions) Methylcellulose, by Nacalai Tesque (1 mentions) Calcein am, by Dojindo Laboratories (1 mentions) Vectashield medium with dapi, by Vector Laboratories (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Bz x710, by Keyence (1 mentions) β tubulin, by Merck Group (1 mentions) Anti human mitochondria antibody, by Abcam (1 mentions) Impact dab, by Vector Laboratories (1 mentions) Ba 1100, by Vector Laboratories (1 mentions) Gill s hematoxylin, by Vector Laboratories (1 mentions)

Close spelling variants of this product

Hybrid Cell Count (18 citations) Hybrid Cell Count Module BZ-H4C (2 citations) BZ-H3C/Hybrid Cell Count Module (2 citations)

8 protocols using hybrid cell count module

Clonogenic Assay of A375/TxR Cells

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In the case of the clonogenic assay, A375/TxR cells were seeded in six-well plates at a very low concentration (1000 cells/well). When each single cell had split into four cells in each well, cells were treated with 5m or 5t at different concentrations (0.5, 1, and 2 nM) or media only and incubated for 7 days. The medium was replaced with the fresh drug once during the treatment. Cells were then fixed with cold methanol and stained with 0.5% crystal violet. Colony area density was quantified using the Keyence Hybrid Cell Count module.

Banerjee S., Mahmud F., Deng S., Ma L., Yun M.K., Fakayode S.O., Arnst K.E., Yang L., Chen H., Wu Z., Lukka P.B., Parmar K., Meibohm B., White S.W., Wang Y., Li W, & Miller D.D. (2021). X-ray Crystallography-Guided Design, Antitumor Efficacy, and QSAR Analysis of Metabolically Stable Cyclopenta-Pyrimidinyl Dihydroquinoxalinone as a Potent Tubulin Polymerization Inhibitor. Journal of medicinal chemistry, 64(17), 13072-13095.

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Quantifying Cartilage Collagen II in 3D and In Vivo

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Sections of in vitro 3D pellets and in vivo tissues were viewed with a BZ‐X800 microscope and then analyzed with BZ‐X800 Analyzer software (KEYENCE). High‐resolution whole images of in vivo tissues were created by seamlessly stitching 30 images at 10× magnification using Multistack Module (KEYENCE). The COL II‐positive area (μm²) was delineated and quantified using Hybrid Cell Count Module (KEYENCE), and the ratio to whole tissue area (WTA) (μm²) was calculated.

Kamakura T., Jin Y., Nishio M., Nagata S., Fukuda M., Sun L., Kawai S, & Toguchida J. (2023). Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC‐Derived Chondrocytes. JBMR Plus, 7(5), e10737.

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SUMO2/3-CnA Protein Interaction Analysis

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Fixed cells were blocked and incubated with two primary antibodies against SUMO2/3 (Abcam, 1:1000, ms) and CnA (Upstate, 1:50, rb). Species-specific probes were added and after annealing and ligation of these probes, an amplification and labeling step was performed that labels probes in close proximity with a green-emitting fluorophore. Green dots within a cell indicate less than 30–40 nm distance between proteins and thus imply a direct protein-protein interaction. Quantitative analysis was carried out utilizing Keyence’s Hybrid Cell Count module as described in detail in supplementary methods. In brief, nuclei were counted and PLA-fluorescence signal extracted to calculate the percentage of positively stained nuclei.

Bernt A., Rangrez A.Y., Eden M., Jungmann A., Katz S., Rohr C., Müller O.J., Katus H.A., Sossalla S.T., Williams T., Ritter O., Frank D, & Frey N. (2016). Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy. Scientific Reports, 6, 35758.

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Quantifying Tumor-Infiltrating Immune Cells

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For the quantification of tumor-infiltrating CD3⁺, CD8⁺, and CD163⁺ immune cell density, 41 images covering 11.04 mm² tissue area were obtained from each CRC slide using an All-in-One Microscope (BZ-X700, KEYENCE; Fig. 1). Four representative region images consisting of 36 views (4 regions × 9 views, total area 9.07 mm²) of the central tumor area were captured, and five representative region images (total area 1.97 mm²) of the invasive margin were captured (Fig. 1B). Tumor-infiltrating CD3⁺, CD8⁺, and CD163⁺ immune cell densities in the central tumor and invasive margins were determined using the Hybrid Cell Count Module (KEYENCE) as a semi-automatic image analysis software. The number of tumor-infiltrating immune cells was divided by the total area (mm²) to calculate the cell density per mm².

Dorjkhorloo G., Erkhem-Ochir B., Shiraishi T., Sohda M., Okami H., Yamaguchi A., Shioi I., Komine C., Nakazawa N., Ozawa N., Shibasaki Y., Okada T., Osone K., Sano A., Sakai M., Ogawa H., Yokobori T., Shirabe K, & Saeki H. (2024). Prognostic value of a modified‑immune scoring system in patients with pathological T4 colorectal cancer. Oncology Letters, 27(3), 104.

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Glioblastoma Spheroid Assay Protocol

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T98G and A172 cells were cultured in spheroid culture medium containing 1.6% (w/v) methylcellulose (Nacalai Tesque). Fresh aliquots of MACS NeuroBrew‐21, EGF and bFGF were added at day 3. After 6 days in culture, the living cells were stained with Calcein‐AM (Dojindo) and counted using hybrid cell count module (Keyence). In some experiments, cells were treated with Strictinin (Nagara Science Co., Ltd.), a Ror1 inhibitor. The numbers and sizes of spheroids formed were measured, and spheroids were classified into small (<1000 μm²), medium (1001–2000 μm²), and large (>2001 μm²) according to their sizes.

Ishikawa T., Ogura Y., Tanaka K., Nagashima H., Sasayama T., Endo M, & Minami Y. (2022). Ror1 is expressed inducibly by Notch and hypoxia signaling and regulates stem cell‐like property of glioblastoma cells. Cancer Science, 114(2), 561-573.

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Immunofluorescent Neuronal Imaging and Analysis

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Following fixation (4% paraformaldehyde), blocking (1% BSA), and washing, neurons were labeled with the following primary antibodies (1:100, overnight, 4 °C): β Tubulin (Sigma; T8578), BAG1 (Santa Cruz; SC376848), BAG3 (Proteintech; 105991AP) MAP2 (Cell Signaling; 4542). Alexa Fluor®secondary antibodies (1:500) (ThermoFisher, Waltham, MA) and VectaShield with DAPI medium (Vector Laboratories, Burlingame, CA) were used for labeling and mounting, respectively. Imaging utilized a fluorescence microscope (BZ-X710; Keyence, Osaka, Osaka, Japan) and image processing employed the Hybrid Cell Count module within the BZ Image Analyzer software (Keyence) to obtain optical density measurements expressed in μM per cell.

Duggan M.R., Mohseni Ahooyi T., Parikh V, & Khalili K. (2021). Neuromodulation of BAG co-chaperones by HIV-1 viral proteins and H2O2: implications for HIV-associated neurological disorders. Cell Death Discovery, 7, 60.

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Quantifying Metastatic Burden in Lung and Liver

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Fixed lung, liver, and tumor tissues were paraffin-embedded and cut into 4 μm thick sections. Histological processing of specimens was carried out by dewaxing, staining with H&E, rehydrating, and sealing to attach to glass slides. Anti-mitochondria IHC staining to visualize metastasis was performed according to previously published protocols.^{12 (link)} Briefly, lung and liver slides were stained with the anti-human mitochondria antibody (AbCAM, Cambridge, MA, Cat# ab92824) with a 1:1000 dilution overnight after blocking with 10% of horse serum. The following day, the slides were incubated with the secondary anti-mouse antibody, visualized with the DAB agent (Sigma-Aldrich, Cat# D5637), counterstained in Gill’s hematoxylin, and mounted with Permount mounting media. Representative tissue images were captured using a Keyence BZ-X700 microscope. Representative whole lung or liver images were digitally scanned using a Pannoramic FLASH III system (3D Histech). Lung or liver metastatic burden of each mouse was quantified by measuring the percentage of the metastasis area in 3–4 representative fields per H&E staining tissue with the Keyence Hybrid Cell Count module.

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Immunohistochemical Analysis of Tumor Samples

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Fixed tumors and organs were embedded in paraffin and unstained slides were prepared for immunostaining or stained with H&E. IHC staining was performed as previously described (21 (link)). Primary antibodies included rabbit anti-Ki67 (1:400), rabbit anti-CD31 (1:100) (#9027; #77699, Cell Signaling Technology), rabbit anti-cleaved PARP (1:50) and rabbit anti-cleaved-caspase-3 (1:200), detected by a biotinylated horse anti-rabbit IgG antibody (BA-1100, Vector Laboratories Inc., Burlingame, CA) secondary antibody. Anti-human-specific mitochondria IHC staining (AbCAM, Cambridge, MA, cat# ab92824) was performed at a 1:1000 dilution to visualize metastases. All slides were developed with DAB Impact (Vector Labs, Burlingame, CA), counterstained in Gill’s hematoxylin (Vector Labs) and mounted with Cytoseal XYL mounting media. Tissue images were acquired with a Keyence BZ-X700 microscope. Quantification of metastatic burden was performed by digital scanning of whole stained slides using a Pannoramic FLASH III system (3D Histech), followed by manual counting of metastatic lesions present in the tissue section. Quantification of Ki67-, CD31-, cleaved-PARP- and cleaved-caspase-3-positive tumor cells was performed by calculating the area of positive cells in 4–5 representative fields per section using the Keyence Hybrid Cell Count module.

Deng S., Krutilina R.I., Wang Q., Lin Z., Parke D.N., Playa H.C., Chen H., Miller D.D., Seagroves T.N, & Li W. (2019). An orally available tubulin inhibitor, VERU-111, suppresses triple-negative breast cancer tumor growth and metastasis and bypasses taxane resistance. Molecular cancer therapeutics, 19(2), 348-363.

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