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Cellinsight cx5 imager

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The CellInsight CX5 imager is a high-content screening (HCS) system designed for automated, quantitative analysis of cellular assays. It captures and analyzes images of cells and provides data on various cellular parameters, such as morphology, fluorescence, and subcellular localization.

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Lab products found in correlation

Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) 96 well plate, by Corning (2 mentions) Cytation 5 cell imaging multi mode reader, by Agilent Technologies (2 mentions) Hoechst, by Thermo Fisher Scientific (1 mentions) Dylight 488, by Thermo Fisher Scientific (1 mentions) Lipofectamine 3000 transfection, by Thermo Fisher Scientific (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions)

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CellInsight (22 citations) CellInsight CX5 (16 citations)

9 protocols using cellinsight cx5 imager

1

Quantification of Pseudovirus Infection

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Vero cells were seeded in 96-well plates at a density sufficient to produce a monolayer at the time of infection. Then, 5-fold serial dilutions of pseudovirus were made and added to cells in triplicate wells. Infection was allowed to proceed for 12–16 h at 37°C. The cells were then fixed with 4% PFA and stained with Hoescht (1μg/mL in PBS). Cells were washed once with PBS and pseudovirus titers were quantified as the number of fluorescent forming units (ffu/mL) using a CellInsight CX5 imager (ThermoScientific) and automated enumeration of cells expressing GFP.
Hastie K.M., Yu X., Ana-Sosa-Batiz F., Zyla D.S., Harkins S.S., Hariharan C., Wasserman H., Zandonatti M.A., Miller R., Maule E., Kim K., Valentine K.M., Shresta S, & Saphire E.O. (2023). Potent Omicron-neutralizing antibodies isolated from a patient vaccinated 6 months before Omicron emergence. Cell Reports, 42(5), 112421.
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2

Quantifying Viral Infectivity and Neutralization

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Viral infectivity was determined by automated counting of eGFP+ cells (infectious units; IU) using a CellInsight CX5 imager (ThermoFisher) at 12–14 h post-infection. For mAb neutralization experiments, pre-titrated amounts of recombinant vesicular stomatitis viruses (rVSVs) expressing eGFP and GP from EBOV, BDBV, TAFV, SUDV, RESTV and BOMV particles (MOI ≈1 IU per cell) were incubated with increasing concentrations of test mAb at room temp for 1 h, and then added to confluent Vero cell monolayers in 96-well plates (Goldstein et al., 2018 (link); Wec et al., 2016 (link); Wong et al., 2010 (link)). Viral neutralization data were subjected to nonlinear regression analysis to derive IC50 values (4-parameter, variable slope sigmoidal dose-response equation; GraphPad Prism, La Jolla, CA).
Wec A.Z., Bornholdt Z.A., He S., Herbert A.S., Goodwin E., Wirchnianski A.S., Gunn B.M., Zhang Z., Zhu W., Liu G., Abelson D.M., Moyer C.L., Jangra R.K., James R.M., Bakken R.R., Bohorova N., Bohorov O., Kim D.H., Pauly M.H., Velasco J., Bortz RH I.I.I., Whaley K.J., Goldstein T., Anthony S.J., Alter G., Walker L.M., Dye J.M., Zeitlin L., Qiu X, & Chandran K. (2019). Development of a human antibody cocktail that deploys multiple functions to confer pan-ebolavirus protection. Cell host & microbe, 25(1), 39-48.e5.
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3

Lassa Virus Glycoprotein Trafficking

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HEK-293T cells seeded in 24-well plates were transfected with phCMV-LASV (lineage IV) GPC. At 24 hours after transfection, cells were fixed or not with 4% PFA for 20 minutes at room temperature before washing with PBS three times. All wells were then blocked with 1% bovine serum albumin in PBS at room temperature for 2 hours. The fixed or unfixed cells were incubated with 8.9F IgG or 37.2D IgG (10 μg/mL) overnight at 4 °C. The cells were then washed twice with PBS, and stained with donkey anti-human IgG DyLight 488 (2 μg/mL, Thermo Fisher Scientific) and Hoechst (10 μg/mL) for 1 hour in the dark at room temperature. Fluorescent images of the stained cells were collected with a CellInsight CX5 imager (Thermo Fisher Scientific) operating in two-channel mode with excitation and emission wavelengths of 386 nm/440 nm and 485 nm/521 nm, respectively.
Eis-Hübinger A.M., Däumer M., Matz B, & Schneweis K.E. (1999). Evaluation of Three Glycoprotein G2-Based Enzyme Immunoassays for Detection of Antibodies to Herpes Simplex Virus Type 2 in Human Sera. Journal of Clinical Microbiology, 37(5), 1242-1246.
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4

Quantitative LASV Virus Neutralization Assay

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Vero cells were seeded in 96-well plates (Corning Life Sciences) at a cell density of 20,000 cells/well in DMEM with 10% FBS and 1% Penn/Strep 5 h prior to infection. Antibodies were diluted to f 0.3 mg/mL (100 nM) and further diluted in a 10-fold dilution series. Each Ab dilution was then mixed 1:1 with a pre-titrated amount of rVSV-LASV-GP and incubated for 1 h at 37°C. Media was removed from the cell monolayer and the Ab/virus mixture was added to duplicate wells and incubated overnight in 5% CO2 at 37°C. Cells were fixed using 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in PBS, and cell nuclei were stained using 1 μg/mL Hoechst-33342. Cells were imaged using a CellInsight CX5 imager (Thermo Fisher) and infection was quantitated by automated enumeration of cells expressing GFP.
Enriquez A.S., Buck T.K., Li H., Norris M.J., Moon-Walker A., Zandonatti M.A., Harkins S.S., Robinson J.E., Branco L.M., Garry R.F., Saphire E.O, & Hastie K.M. (2022). Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies. Cell reports, 39(8), 110841.
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5

Viral Infectivity Quantification and Antibody Neutralization Assay

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Viral infectivities were measured by automated counting of eGFP+ cells (infectious units; IU) using a CellInsight CX5 imager (Thermo Fisher) at 12–14 hr post-infection. For mAb neutralization experiments, pre-titrated amounts of VSV-GP particles (MOI ≈ 1 IU per cell) were incubated with increasing concentrations of test mAb at room temp for 1 hr, and then added to confluent cell monolayers in 96-well plates. Viral neutralization data were subjected to nonlinear regression analysis to derive EC50 values (4-parameter, variable slope sigmoidal dose-response equation; GraphPad Prism).
Wec A.Z., Herbert A.S., Murin C.D., Nyakatura E.K., Abelson D.M., Fels J.M., He S., James R.M., de La Vega M.A., Zhu W., Bakken R.R., Goodwin E., Turner H.L., Jangra R.K., Zeitlin L., Qiu X., Lai J.R., Walker L.M., Ward A.B., Dye J.M., Chandran K, & Bornholdt Z.A. (2017). Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses. Cell, 169(5), 878-890.e15.
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6

Quantitative LASV Virus Neutralization Assay

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Vero cells were seeded in 96-well plates (Corning Life Sciences) at a cell density of 20,000 cells/well in DMEM with 10% FBS and 1% Penn/Strep 5 h prior to infection. Antibodies were diluted to f 0.3 mg/mL (100 nM) and further diluted in a 10-fold dilution series. Each Ab dilution was then mixed 1:1 with a pre-titrated amount of rVSV-LASV-GP and incubated for 1 h at 37°C. Media was removed from the cell monolayer and the Ab/virus mixture was added to duplicate wells and incubated overnight in 5% CO2 at 37°C. Cells were fixed using 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in PBS, and cell nuclei were stained using 1 μg/mL Hoechst-33342. Cells were imaged using a CellInsight CX5 imager (Thermo Fisher) and infection was quantitated by automated enumeration of cells expressing GFP.
Enriquez A.S., Buck T.K., Li H., Norris M.J., Moon-Walker A., Zandonatti M.A., Harkins S.S., Robinson J.E., Branco L.M., Garry R.F., Saphire E.O, & Hastie K.M. (2022). Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies. Cell reports, 39(8), 110841.
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7

Pseudotyped VSV Infection Assay

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Confluent U2OS cells were infected with pre-titrated amounts of pseudotyped VSV particles bearing wild-type or mutant GPs. Prior to infection, VSVs were diluted in corresponding media, and infected cells were maintained at 37°C for 14 to 16 h post-infection before manual counting of eGFP+ and mNG+ cells or automated counting using a Cytation 5 cell imaging multimode reader (BioTek Instruments) and a CellInsight CX5 imager (Thermo Fisher) including onboard software. When indicated, cells were preincubated with small-molecule inhibitors diluted in corresponding media; for antibody neutralization experiments, VSV particles were incubated with increasing concentrations of test antibodies at room temperature for 1 h prior to addition to cell monolayers. Virus infectivities were measured as described above. Virus neutralization data were subjected to nonlinear regression analysis (4-parameter, variable slope sigmoidal dose-response equation; GraphPad Prism).
Mittler E., Alkutkar T., Jangra R.K, & Chandran K. (2021). Direct Intracellular Visualization of Ebola Virus-Receptor Interaction by In Situ Proximity Ligation. mBio, 12(1), e03100-20.
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8

Generation of EBOV-Pseudotyped VSV-ΔG-GFP

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Recombinant EBOV-pseudotyped VSV-ΔG-GFP were generated by transfecting 293T cells with phCMV3-EBOV-GP using Lipofectamine 3000 transfection reagent (ThermoFisher) according to the manufacturer’s instructions. At 24 h post-transfection, cells were washed 2x with Opti-MEM (Gibco) and infected with rVSV-G pseudotyped DG-GFP parent virus (VSV-G*ΔG-GFP) at MOI = 3 for 1 h with rocking every 15 min. The virus was then removed, and the cells were washed twice with Opti-MEM containing 2% FBS (Gibco) before fresh media was added. Supernatants containing rVSV-SARS-2 were removed 24 h post-infection and clarified by centrifugation.
Vero cells were seeded in 96-well plates at a density sufficient to produce a monolayer at the time of infection. Then, 10-fold serial dilutions of pseudovirus were made and added to cells in triplicate wells. Infection was allowed to proceed for 20 h at 37°C. The cells were then fixed with 4% paraformaldehyde (PFA), washed with 1xPBS and stained with Hoechst (2 μg/mL in PBS). PBS (50 μL) was added after removal of stain for imaging. Pseudovirus titers were quantified as the number of fluorescent forming units (ffu/mL) using a CellInsight CX5 imager (ThermoScientific) and automated enumeration of cells expressing GFP.
Yu X., Hastie K.M., Davis C.W., Avalos R.D., Williams D., Parekh D., Hui S., Mann C., Hariharan C., Takada A., Ahmed R, & Saphire E.O. (2023). The evolution and determinants of neutralization of potent head-binding antibodies against Ebola virus. Cell reports, 42(11), 113366.
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9

Pseudotyped VSV Hantavirus Neutralization Assay

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Confluent Vero cells were inoculated with pre-titrated amounts of pseudotyped rVSV particles bearing either PUUV, HTNV, SEOV, DOBV, ANDV, SNV, and CHOV Gn/Gc or, when indicated, rVSV-PUUV-Gn/Gc neutralization-escape mutants. Prior to infection, rVSVs were diluted in corresponding media, and infected cells were maintained at 37°C for 14 to 16 h post-infection before automated counting of eGFP+ and mNG+ cells using a Cytation 5 cell imaging multi-mode reader (BioTek Instruments) or a Cell-Insight CX5 imager (Thermo Fisher) including onboard software. For neutralization experiments, rVSV particles were incubated with increasing concentrations of mAbs or, when indicated, human serum diluted in corresponding media at room temperature for 1 h prior to addition to cell monolayers. Virus neutralization data were subjected to nonlinear regression analysis to derive IC50 values (4-parameter, variable slope sigmoidal dose-response equation constraining bottom parameters to 0-100; GraphPad Prism).
Mittler E., Wec A.Z., Tynell J., Guardado-Calvo P., Wigren-Byström J., Polanco L.C., O’Brien C.M., Slough M.M., Abelson D.M., Serris A., Sakharkar M., Pehau-Arnaudet G., Bakken R.R., Geoghegan J.C., Jangra R.K., Keller M., Zeitlin L., Vapalahti O., Ulrich R.G., Bornholdt Z.A., Ahlm C., Rey F.A., Dye J.M., Bradfute S.B., Strandin T., Herbert A.S., Forsell M.N., Walker L.M, & Chandran K. (2022). Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses. Science translational medicine, 14(636), eabl5399.
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