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Cytation 5 imaging system

Manufactured by Agilent Technologies
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The Cytation 5 imaging system is a multi-mode microplate reader and cell imager that combines automated digital microscopy and microplate detection capabilities in a single, compact benchtop instrument. It provides high-performance imaging and quantitative analysis for a wide range of cell-based and molecular assays.

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Lab products found in correlation

Rm2255 microtome, by Leica (2 mentions) Gen5 3, by Agilent Technologies (1 mentions) Scn400 slide scanner, by Leica (1 mentions) F actin, by Thermo Fisher Scientific (1 mentions) Superfrost glass slides, by Leica (1 mentions) Seahorse wave desktop software, by Agilent Technologies (1 mentions) Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions) Fluosphere, by Thermo Fisher Scientific (1 mentions) Dopamine hydrochloride, by Merck Group (1 mentions) Formalin, by Merck Group (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) Agarose, by Merck Group (1 mentions) Axiovert 200m microscope, by Zeiss (1 mentions)

15 protocols using cytation 5 imaging system

1

Multiparametric Immunocytochemical Characterization

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The cells were fixed using 4% paraformaldehyde and blocked with phosphate-buffered saline with Tween 20 solution that contained bovine serum albumin and glycine. Differentiated cells were then analyzed by immunocytochemical staining with primary antibodies mouse anti-RIP (1:200), rabbit anti-nestin (1:200), rabbit anti-MBP (1:200), mouse anti-FAK (1:200), and rabbit anti-GFAP (1:200), followed by goat anti-mouse Alexa Fluor 488 (green) and goat anti-rabbit Alexa Fluor 594 (red) conjugated secondary antibodies. Lastly, the cells were counterstained with DAPI to stain the nuclei (blue).
Immunolabeled cells were scanned (22 × 20 montage at 10× magnification for each well) using the Cytation5 imaging system (BioTek, Santa Clara, CA, USA), and cells were quantified in an unbiased manner using Gen5 3.05 Software (BioTek). DAPI staining was used to determine the total cell count.
Sharma K.D., Alghazali K.M., Hamzah R.N., Pandanaboina S.C., Nima Alsudani Z.A., Muhi M., Watanabe F., Zhou G.L., Biris A.S, & Xie J.Y. (2022). Gold Nanorod Substrate for Rat Fetal Neural Stem Cell Differentiation into Oligodendrocytes. Nanomaterials, 12(6), 929.
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2

Osteoclast Quantification in Distal Femur Metaphysis

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Following microCT scanning, femurs were analyzed for osteoclast abundance in the distal metaphyseal trabecular bone using histomorphometry, as done previously (Putnam et al., 2019 (link)). Femurs were decalcified in 20% EDTA for 3 days, paraffin embedded, and sectioned on a Leica RM2255 microtome (Leica Biosystems, Buffalo Grove, IL, USA) at a thickness of 4 μm. Sections were stained for tartrate-resistant acid phosphatase (TRAP) with hematoxylin counterstain. TRAP-stained slides were imaged with a Leica SCN400 Slide Scanner (Leica Biosystems) in brightfield at 20X or on a Cytation 5 Imaging System [BioTek Instruments, Winooski, VT, USA] in brightfield at 10X and analyzed with Bioquant software (Bioquant Image Analysis Corporation, Nashville, TN, USA). Bioquant software was used to measure osteoclast number, osteoclast surface, and bone surface in the trabeculae of the distal metaphysis of the femur proximal to the growth plate on the TRAP-stained slides. According to ASBMR standards (Dempster et al., 2013 (link)), osteoclast number, osteoclast surface, and bone surface were measured to calculate osteoclast number per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS).
Ford C.A., Hurford I.M., Fulbright L.E., Curry J.M., Peek C.T., Spoonmore T.J., Cruz Victorio V., Johnson J.R., Peck S.H, & Cassat J.E. (2022). Loss of Vhl alters trabecular bone loss during S. aureus osteomyelitis in a cell-specific manner. Frontiers in Cellular and Infection Microbiology, 12, 985467.
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3

Quantitative Live Cell Imaging for Proliferation

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Cells were cultured under conditions as described in the preceding section in 96 well plates. Live cell imaging was performed using a Cytation 5 imaging system (Biotek) using a 4x objective. High contrast brightfield imaging for label free live cell proliferation measurements was accomplished by capturing two high contrast brightfield images at each time point, one in focus image for reference and a defocused image, with an offset of −250 μm from the in focal plane, for cell counting. Images were processed with Gen 5 to get total cell counts as well as counts of mitotic cells which have a relatively high circularity compared with cells in other cell cycle phases. For cellular analysis the defocused brightfield image was processed using a dark background setting and a flattening size of 10 μm. A primary mask was applied using a threshold of 5,000 relative intensity units along with background percentage of 5%, a minimum object size of 5 μm and a maximum object size of 50 μm. Identification of mitotic cells was accomplished by applying a secondary mask to the images, selecting for objects with a circularity of 0.5 or greater with an area of less than 500 as well as an intensity of 18000 or more.
Chavez J.D., Keller A., Zhou B., Tian R, & Bruce J.E. (2019). Cellular Interactome Dynamics during Paclitaxel Treatment. Cell reports, 29(8), 2371-2383.e5.
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4

Quantitative Live Cell Imaging for Proliferation

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Cells were cultured under conditions as described in the preceding section in 96 well plates. Live cell imaging was performed using a Cytation 5 imaging system (Biotek) using a 4x objective. High contrast brightfield imaging for label free live cell proliferation measurements was accomplished by capturing two high contrast brightfield images at each time point, one in focus image for reference and a defocused image, with an offset of −250 μm from the in focal plane, for cell counting. Images were processed with Gen 5 to get total cell counts as well as counts of mitotic cells which have a relatively high circularity compared with cells in other cell cycle phases. For cellular analysis the defocused brightfield image was processed using a dark background setting and a flattening size of 10 μm. A primary mask was applied using a threshold of 5,000 relative intensity units along with background percentage of 5%, a minimum object size of 5 μm and a maximum object size of 50 μm. Identification of mitotic cells was accomplished by applying a secondary mask to the images, selecting for objects with a circularity of 0.5 or greater with an area of less than 500 as well as an intensity of 18000 or more.
Chavez J.D., Keller A., Zhou B., Tian R, & Bruce J.E. (2019). Cellular Interactome Dynamics during Paclitaxel Treatment. Cell reports, 29(8), 2371-2383.e5.
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5

Preparation and Titration of RVPs

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RVPs were prepared by transfecting 293 T cells with WNV Rep/GFP replicon construct along with WNV or ZIKV or DENV-1 CprME expression constructs. The RVPs were harvested 48 h post transfection, aliquoted and stored for future use. RVPs were titrated in Vero cells plated in 96 well clear bottom black plates at 5000 cells per well. Serial two-fold dilutions of the RVPs were used to infect Vero cells and incubated for 72 h. The plates were fixed using 4% formalin in PBS and images acquired using the Cytation5 imaging system (BioTek, Winooski, VT, USA). Images of whole wells were acquired and the number of GFP+ cells per well was quantified using Gen5 software (BioTek).
Garg H., Yeh R., Watts D.M., Mehmetoglu-Gurbuz T., Resendes R., Parsons B., Gonzales F, & Joshi A. (2021). Enhancement of Zika virus infection by antibodies from West Nile virus seropositive individuals with no history of clinical infection. BMC Immunology, 22, 5.
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6

Monitoring miR-145 Expression in Cells

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The pCMV vectors encode a green fluorescent protein (GFP) for monitoring transfection efficiency. Cytation 5 imaging system (Cytation 5, Biotek, Winooski, VT) was employed to detect the GFP-expressing cells. The GFP+ cells were evaluated at 475 nm. Then, 48 h later, the cells were examined using flow cytometry. In brief, MKN-45 cells transfected with pCMV-miR-145 or blank vector were suspended in phosphate-buffered saline (PBS) at 1× 106 cells/ml. Stable cells were generated over two weeks of the selection time in the presence of 4 μg/μl Geneticin (G418, Gibco). The stable cells were evaluated by flow cytometry (Miltenyi Biotec, Germany). Concisely, the transfected cells emitted the green fluorescence due to the expression of GFP. Consequently, the portion of the GFP positive cells was evaluated in the FITC channel, and the expression of miR-145 was measured by qRT-PCR.
Zeinali T., Karimi L., Hosseinahli N., Shanehbandi D., Mansoori B., Mohammadi A., Hajiasgharzadeh K., Babaloo Z., Majidi-Zolbanin J, & Baradaran B. (2020). Overexpression of miRNA-145 induces apoptosis and prevents proliferation and migration of MKN-45 gastric cancer cells. EXCLI Journal, 19, 1446-1458.
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7

Wound Closure Kinetics of RPE-1 Cells

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RPE-1 cells were plated confluent in a 12-well dish (106 cells per well). The next day a wound was performed using a pipet tip. After a wash with complete medium, the closure of the wound was followed overtime by phase contrast video-microscopy using the Cytation 5 imaging system (Biotek, Colmar, France). Images were taken every 15 min and the area of the wound closure calculated using the software ImageJ. The graph represents the percentage of closure relative to the surface area of the wound at t = 0 from 3 independent experiments performed in triplicates ± SD.
Macia E., Vazquez-Rojas M., Robiolo A., Fayad R., Abélanet S., Mus-Veteau I., Fontaine-Vive F., Mehiri M., Luton F, & Franco M. (2021). Chlortetracycline, a Novel Arf Inhibitor That Decreases the Arf6-Dependent Invasive Properties of Breast Cancer Cells. Molecules, 26(4), 969.
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8

Osteoclast Formation from Bone Marrow

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FACS purified populations were used immediately after sorting. 5.0 × 105 unsorted whole bone marrow or 1.0 × 104 FACS purified populations were seeded per well into 96-well plates supplemented with 5% v/v CMG14-12 supernatant (as a source of M-CSF) and 35 ng/mL RANKL. Cells were cultured for 4 days, at which point the cells were fixed and stained for TRAP per the manufacturer’s instructions (Sigma). Where indicated, cells were treated with 10 μg/mL of either anti-MDL-1 (R&D, clone 226402) or IgG2a isotype control (R&D clone 54447). Fixed cells were stained with DAPI and imaged using a Cytation 5 imaging system (Biotek) prior to manual enumeration of multi-nucleated TRAP+ cells as osteoclasts.
Peek C.T., Ford C.A., Eichelberger K.R., Jacobse J., Torres T.P., Maseda D., Latour Y.L., Piazuelo M.B., Johnson J.R., Byndloss M.X., Wilson K.T., Rathmell J.C., Goettel J.A, & Cassat J.E. (2022). Intestinal Inflammation Promotes MDL-1+ Osteoclast Precursor Expansion to Trigger Osteoclastogenesis and Bone Loss. Cellular and Molecular Gastroenterology and Hepatology, 14(4), 731-750.
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9

Immunofluorescence Analysis of MSCs

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MSCs were seeded onto sterile coverslips and allowed to grow till 60% confluency. The plated cells were fixed with 4% paraformaldehyde and permeabilized using 0.2% Triton X in PBS at RT. The cells were then stained with respective primary and secondary antibodies, and phalloidin (for F-actin, Invitrogen). Thereafter, the cells were counter stained with DAPI (4′,6-diamidino-2-phenylindole) for nuclei. The cells were imaged using Cytation 5 imaging system (BioTek Instruments).
Abu-El-Rub E., Sequiera G.L., Sareen N., Yan W., Moudgil M., Sabbir M.G, & Dhingra S. (2019). Hypoxia-induced 26S proteasome dysfunction increases immunogenicity of mesenchymal stem cells. Cell Death & Disease, 10(2), 90.
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10

Quantitative Histomorphometry of Osteoclasts

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Following micro CT imaging, femurs were decalcified for 3 days in 20% EDTA (pH 7.4) at 4 ºC. Samples were then dehydrated, embedded in paraffin blocks, and sectioned longitudinally at 4 μm thickness through the medullary cavity with a Leica RM2255 microtome. Tissue sections were mounted onto Leica Superfrost glass slides and then stained with TRAP stain with hematoxylin counterstain. Bioquant software (Nashville, TN) was used to perform quantitative histomorphometry (osteoclast number, osteoclast surface, and bone perimeter) in accordance with ASBMR guidelines using 10× images generated from a Cytation 5 imaging system (Biotek).69 (link)
Peek C.T., Ford C.A., Eichelberger K.R., Jacobse J., Torres T.P., Maseda D., Latour Y.L., Piazuelo M.B., Johnson J.R., Byndloss M.X., Wilson K.T., Rathmell J.C., Goettel J.A, & Cassat J.E. (2022). Intestinal Inflammation Promotes MDL-1+ Osteoclast Precursor Expansion to Trigger Osteoclastogenesis and Bone Loss. Cellular and Molecular Gastroenterology and Hepatology, 14(4), 731-750.
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