The invasiveness of the cultured cells was assessed by the number of migrating cells through Matrigel-coated Transwell inserts, as described previously.26 (link) The upper surface of a filter (pore size, 8.0 μm; BD Biosciences, Heidelberg, Germany) was coated with basement membrane Matrigel (BD Biosciences). Cells (1.0 × 104) were re-plated to the upper chamber and incubated in a medium for 16 h. The total number of cells that had migrated to the lower side of the filter was counted using a BZ-9000 (Biorevo; Keyence, Osaka, Japan) and Hybrid cell count software (Keyence).
Hybrid cell count software
Manufactured by Keyence
Sourced in Japan
The Hybrid Cell Count software is a tool designed to analyze and quantify cell samples. It utilizes a combination of image processing and automated counting algorithms to provide accurate cell counts. The software is capable of handling a variety of cell types and sample preparations, making it a versatile tool for laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Bz 9000 fluorescence microscope, by Keyence (3 mentions)
Bz x710 microscope, by Keyence (3 mentions)
Bz x800, by Keyence (2 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Bz x810, by Keyence (2 mentions)
Bz x700 fluorescence microscope, by Keyence (2 mentions)
Bz x700 microscope, by Keyence (2 mentions)
Matrigel, by BD (1 mentions)
Duolink kit, by Merck Group (1 mentions)
Goat anti rabbit igg alexa 568, by Thermo Fisher Scientific (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Ncl spec1, by Leica (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
Bz x analysis software, by Keyence (1 mentions)
Bz x810 system, by Keyence (1 mentions)
Aquacosmos 2, by Hamamatsu Photonics (1 mentions)
Alexa488 labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bz x710, by Keyence (1 mentions)
38 protocols using hybrid cell count software
1
Transwell Invasion Assay Protocol
2
PLA Experiments for RAD51-FANCD2 and RAD51-PCNA
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PLA experiments were performed according to the manufacturer's protocol, using a DuoLink Kit (Sigma-Aldrich). For the PLA experiments between RAD51 and FLAG-tagged FANCD2, cells were treated with 100 ng/ml MMC or 0.5 mM HU (or not treated) for 24 h, and were fixed with 2% sucrose, 3% bovine serum albumin (BSA) and 0.5% Triton X-100 in PBS, followed by staining with an anti-FLAG tag (anti-DDDDK-tag) mouse Ab (1:500; PM020, MBL) and an anti-RAD51 rabbit Ab (1:500). For the PLA experiments between RAD51 and PCNA, cells were transfected with the control siRNA or the FANCD2 siRNA, and were fixed at 48 h after transfection. The HU treatment (4 mM) was performed for 2.5 h immediately before sample fixation. The cells were then stained with an anti-PCNA mouse Ab (1:500; PC10, Santa Cruz Biotechnology, Inc.) and an anti-RAD51 rabbit Ab (1:500). PLA signals were detected with a fluorescence microscope (BZ-9000), and nuclear PLA signals were analyzed by counting the red signals on DAPI-stained blue areas, using the hybrid cell count software (KEYENCE). In total, >50 cells were analyzed, and were plotted in each column. Statistical differences were determined by the Student's t-test with the Prism software.
3
Quantifying CD8+ Lymphocytes in Primary Tumor
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The tissue slide evaluated by IHC was made from primary lesions not metastatic lesions. The counting method for CD8+ lymphocytes in tissue was as follows: in a single slide including the primary tumor, nine areas with many CD8+ lymphocytes at invasive front were chosen (Figure 3A ). The images were divided into three equal parts: tissue, boundary, and stroma parts (Figure 3B , ×200). CD8+ lymphocytes within the area (0.104 mm2 in each area) were detected by fluorescence immunostaining, and the number of cells was calculated using Hybrid Cell Count software (BZ‐X800; Keyence, Figure 3C ), as previously reported.31 The maximum number of CD8+ lymphocytes among the nine areas measured by the hybrid cell count was defined as the CD8+ lymphocyte count in the tissue.
4
Quantifying CD8+ Lymphocyte Density
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The number of CD8+ lymphocytes which did not form cell cluster in the background of cell block was counted as similar method to count the number of CD8+ lymphocyte in the tissue. Briefly, nine sites without tumor cell clusters were randomly selected. The number of CD8+ and FoxP3+ lymphocytes were counted using Hybrid Cell Count software (BZ‐X800; Keyence).
5
Histological Analysis of Lung Tissues
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The lungs were dissected and fixed by soaking the tissues in phosphate buffered saline (PBS) containing 4% paraformaldehyde. The fixed lung tissues were embedded in paraffin for sectioning (5 μm-thick) and stained with hematoxylin and eosin (HE). The specimens were observed and analyzed using a microscope BZ-X800 and a hybrid cell count software (Keyence, Osaka, Japan).
6
Immunofluorescence Analysis of Muscle Cytoskeleton
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Cross-sections of TA muscle were fixed in cold acetone for 10 min and air-dried. After blocking with 5% goat serum and 2% BSA in PBS(-), tissue sections were incubated with antibodies to human lamin A/C (mouse monoclonal, NCL-LAM-A/C, Leica), human spectrin (mouse monoclonal, NCL-SPEC1, Leica), and dystrophin (rabbit polyclonal, Abcam) (100–400X dilution) overnight at 4 °C. The next day, primary antibodies were washed out with PBS(-), then incubated with Alexa 488 goat-anti-mouse IgG2b, Alexa568 goat anti-rabbit IgG, or Alexa568-goat anti-mouse IgG2aκ (Molecular Probes) for 2 h, and mounted in Vecta Shield containing DAPI (Vector). Images were recorded with a KEYENCE BZ-9000 microscope and analyzed using hybrid cell count software (Keyence Corp.) or Image J.
7
Quantification of RNA-DNA Hybrid Foci
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Cells cultured on 15-mm coverslips were washed with PBS and fixed with PBS containing 3% paraformaldehyde, 2% sucrose, 0.5% Triton-X-100, placed on ice for 30 min, and further permeabilized with 0.5% Triton X-100/PBS for 5 min. After blocking with 2% BSA/PBS, cells were stained with the indicated primary antibody for 1 hr at RT. For quantification of the S9.6 foci, cells were pre-extracted with 0.5% Triton X-100/PBS for 5 min, fixed with 4% paraformaldehyde for 10 min, and further extracted with 100% cold methanol at -20℃ for 5 min. Slides were blocked with 2% BSA/PBS, then stained with S9.6 antibodies for 1 hr at RT. Alexa Fluor 488-conjugated anti-mouse IgG or Alexa Fluor 594-conjugated anti-rabbit IgG (Molecular Probes) were used as secondary antibodies. PLA was performed with reagents from DuoLink Biosciences, which were used according to the manufacturer's instructions. Images were captured using a BZ-9000 fluorescence microscope (Keyence). Quantification of the PLA signal dots and FANCD2 foci was carried out using Hybrid cell count software (Keyence). The number of S9.6 foci were enumerated using an IN Cell Analyzer 2000 (GE Healthcare). Briefly, the nuclear areas were defined by DAPI staining and then anti-nucleolin positive areas (corresponding to nucleoli) were subtracted. The nuclear S9.6 foci number was determined in the remaining subnuclear areas.
8
Immunofluorescence and Proximity Ligation Assay
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Cells were cultured on 15-mm coverslips and indicated plasmids were transfected. Cells on coverslips were washed with PBS twice and fixed with PBS containing 3% paraformaldehyde, 2% sucrose, 0.5% Triton-X-100, chilled on ice for 30 min, and then permeabilized with 0.5% Triton X-100/PBS for 5 min. After blocking with 2% BSA/PBS, cells were stained with the indicated primary antibody diluted in 2% BSA/PBS for 1 h at RT. The secondary antibodies used were Alexa Fluor 488-conjugated anti-mouse IgG or Alexa Fluor 594-conjugated anti-rabbit IgG (Molecular Probes). PLA was performed with reagents from DuoLink Biosciences in accordance with the manufacturer's instructions. Images were captured using a BZ-9000 fluorescence microscope (Keyence). Quantification of the PLA signal dots and FANCD2 or RPA foci was determined using Hybrid cell count software (Keyence).
9
Soft Agar Assay for Anchorage-Independent Growth
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Anchorage-independent growth of sorted cells was evaluated by soft agar colony formation assay. The sorted cells were passaged within 2 weeks before processing. The cells (1 × 104 cells) were incubated in a top layer of 0.4% noble agar (Difco, catalog no. 214220) in DMEM with 10% FBS. The suspension was overlaid on a bottom layer of 0.6% noble agar in DMEM with 10% FBS in a 6-well plate. DMEM was added to each well and cultured at 37°C for 2 to 4 weeks. Each well was scanned automatically using the BZ-X810 system (Keyence), and the whole well was reconstructed from the scanned images using the BZ-X Analysis software (Keyence). Colonies larger than 20,000 μm2 were counted using the Hybrid Cell Count software (Keyence). The experiments were conducted three times independently.
10
Myogenic Differentiation Evaluation Protocol
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Myogenic differentiation was evaluated with immunocytostaining for myosin heavy chain (MF20, R&D Systems), MYOGENIN (Santa Cruz Biotechnology, FD5 or rabbit polyclonal), Pax7 (Santa Cruz Biotechnology, PAX7), and MyoD (5.8 A or rabbit polyclonal; Santa Cruz Biotechnology). Cells were fixed in 4% paraformaldehyde and then permeated with 0.1% Triton-X100 for 10 min at room temperature. After blocking with 5% goat serum/2% bovine serum albumin in PBS(−), cells were incubated with primary antibodies (100–400 dilution) overnight at 4 °C. The next day, the cells were washed in PBS(−) and incubated with fluorescence-labeled secondary antibodies (Alexa 568-labeled goat-anti-mouse IgG2b or Alexa488-labeled goat anti-rabbit IgG)(Molecular Probes) for 2 h. After washing with PBS(−), the nuclei were stained with Hoechst 33258 (Dojindo). The images were recorded using a KEYENCE BZX-710 and analyzed with hybrid cell count software (Keyence Corp.) or IX71 (Olympus) equipped with an ORCA-R2 digital CCD camera and AQUACOSMOS2.6 software (Hamamatsu Photonics).
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