Gen5 3

Manufactured by Agilent Technologies

Sourced in United States

GEN5 3.0 software is a data analysis and visualization tool designed for use with Agilent's microplate readers. The software provides users with tools to analyze, interpret, and present data generated from experiments conducted on Agilent's microplate readers.

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51 protocols using gen5 3

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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Quantifying Nascent Protein Synthesis

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Nascent global protein synthesis was monitored by Protein Synthesis Assay Kit (Caymen Chemicals; Ann Arbor, MI, USA) per manufacturer’s protocol. In short, OCI-LY-10 and OCI-LY-3 were plated in 96-well black plate (Corning, Kennebunk, ME) at 200,000 cells per well and incubated overnight. Cells were pretreated with control (DMSO), CEAA (1 μM), DeBAA (1 μM), AA (1 μM), or Cycloheximide (1 μM) for 1 h; O-Propargyl-puromycin (OPP) was added and incorporated into newly synthesized protein for 2 h. Cells were fixed for 5 min, washed three times, and stained with 5-Fam-Azide solution for 30 min. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Dapi), 1 µg/mL (Thermo Scientific; Rockford, IL, USA). Cells were resuspended in 1 X Tris-buffered saline (TBS) after three additional washes and cells were imaged with BioTek’s Cytation^TM 5 Cell Imaging Multi-Mode Reader (ex:490/ex:525) using Gen 5 3.05 software (BioTek; Winooski, VT, USA). Average background (no OPP) RFU was subtracted from RFU of samples (plated in triplicate) and results were normalized to control.

Caulfield T.R., Hayes K.E., Qiu Y., Coban M., Seok Oh J., Lane A.L., Yoshimitsu T., Hazlehurst L., Copland J.A, & Tun H.W. (2020). A Virtual Screening Platform Identifies Chloroethylagelastatin A as a Potential Ribosomal Inhibitor. Biomolecules, 10(10), 1407.

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Quantifying Ki67 Positive Cells

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A graphic representation of this process is provided in Supplementary Figure 7A. Ki67 and DAPI-stained cells were imaged with a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Using the Gen5 3.05 software (BioTek), the DAPI channel was used to count the number of cells and create an individual mask on the area of each nucleus. Ki67 intensity within each mask was then quantified. A Ki67 threshold intensity of greater than 10,000 was deemed positive. Percentage was determined for each well by dividing the number of positive cells over the total number of counted cells.

DiGiacomo J.W., Godet I., Trautmann-Rodriguez M, & Gilkes D.M. (2020). Extracellular matrix-bound FGF2 mediates estrogen receptor signaling and therapeutic response in breast cancer. Molecular cancer research : MCR, 19(1), 136-149.

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Visualizing Lipid Droplets in Macrophages

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MPMs were cultured on a coverslip for 1 day and treated with acLDL (50 μg/mL) or Hi-oxLDL acLDL (50 μg/mL) for 24h (n = 3 per treatment). Untreated MPMs were used as a control. The cells were fixed with 3.7% P-formaldehyde for 15min at R.T. After washing with 1× PBS three times, the cells were stained with Bodipy 493/503 (5–10 μg/mL) to visualize lipid droplets (green) and DAPI (0.5–1 μg/mL) for nuclei staining (blue) at R.T for 30min. Cells were mounted with ProLong antifade mountant. Images were taken with Zeiss Observer D1 microscope and Cytation 5 imaging reader (BioTek, Winooski, VT, USA). Pictures of three randomly chosen areas per coverslip were analyzed with Gen5 3.05 software (BioTek, Winooski, VT, USA). Each area contained 50–70 cells. Total area of LDs and total fluorescence intensities per cells were obtained using Gen5 3.05 software.

Paul A., Lydic T.A., Hogan R, & Goo Y.H. (2019). Cholesterol Acceptors Regulate the Lipidome of Macrophage Foam Cells. International Journal of Molecular Sciences, 20(15), 3784.

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Brain Penetration of Evans Blue Tracer

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A total of 4% Evan’s Blue (Sigma Aldrich, St. Louis, MO, USA) solution made in 0.9% NaCl (saline; Baxter, Deerfield, IL, USA) was infused into the jugular vein of an anesthetized rat and circulated for 30 min followed by a saline flush. The brain was dissected into the following four regions: frontal cortex (region above the anterior of the circle of Willis), posterior cortex, brainstem, and cerebellum. These four regions were selected as patients with CKD had evidence of impairment in these regions [42 (link),43 (link),44 (link)]. The brain regions were weighed and homogenized in trichloroacetic acid (TCA; Fisher Scientific, Fair Jawn, NJ, USA) buffer solution (1:1 20% w/v TCA and 0.9% saline) at a 1:3 dilution (w/v) as previously described [45 (link)]. The homogenized samples were centrifuged for 20 min at 10,000× g. The supernatant was assayed in triplicates, and samples and standards received 95% ethanol. The Evan’s Blue concentration was measured immediately at an excitation of 620 nm and an emission of 720 nm using Synergy LX (BioTek, Santa Clara, CA, USA) and Gen5 3.05 software (Biotek, Santa Clara, CA, USA). Data are presented as concentration of Evan’s Blue (pg)/weight of tissue (g).

Griffin A., Berry B., Spencer S.K., Bowles T, & Wallace K. (2023). Indoxyl Sulfate Administration during Pregnancy Contributes to Renal Injury and Increased Blood–Brain Barrier Permeability. International Journal of Molecular Sciences, 24(15), 11968.

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Quantifying Cellular Ki67 Expression

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Ki67 and DAPI-stained cells were imaged in a 3 by 3 montage per well with a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Using the Gen5 3.05 software (BioTek), the DAPI channel was used to count the number of cells in a field of view and to create an individual mask on the area of each nucleus. Ki67 intensity within each individual mask was then quantified. By visually observing cells, a threshold intensity of 8,000 was selected. Any object with an intensity greater or equal to the threshold was deemed positive, and all others were deemed negative. Percentage was determined for each well by dividing the number of positive cells over the total number of counted cells.

Mamo M., Ye I.C., DiGiacomo J.W., Park J.Y., Downs B, & Gilkes D.M. (2020). Hypoxia alters the response to anti-EGFR therapy by regulating EGFR expression and downstream signaling in a DNA methylation-specific and HIF-dependent manner. Cancer research, 80(22), 4998-5010.

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Spheroid Growth Kinetic Analysis

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Each spheroid of the microplates was automatically imaged using bright field microscopy module of the Cytation™3MV cell analyzer (Biotek^®-M = 4X) coupled with Gen5 3.08 software (Biotek^®, Winooski, VT, USA). The “Cellular analysis” tool was applied to a stitched picture to calculate the object size of each well (threshold = 20,000, background = light, min. object size = 150 µm, max. object size = 1000 µm). Spheroid size was recorded for each condition on the 1st, 3rd, 4th, 7th, 10th, and 14th days of culture (D1, D3, D4, D7, D10 and D14) allowing growth kinetic curves to be set. Growth curve slopes were calculated for each spheroid condition between the 4th and the 14th culture days (corresponding to the poststorage period) [26 (link)].

Goisnard A., Dubois C., Daumar P., Aubel C., Depresle M., Gauthier J., Vidalinc B., Penault-Llorca F., Mounetou E, & Bamdad M. (2021). The New Serum-Free OptiPASS® Medium in Cold and Oxygen-Free Conditions: An Innovative Conservation Method for the Preservation of MDA-MB-231 Triple Negative Breast Cancer Spheroids. Cancers, 13(8), 1945.

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Fluorescent Imaging of Cell Lines

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For the adherent SUM1315 cell line, cells were seeded in IbiTreat 8-well μ-slides (Ibidi^®, Gräfelfing, Germany) at a concentration of 50 × 10³ cells per well. Slides were maintained for 3 days at 37 °C in a humidified incubator in order to obtain homogeneous confluent culture. The suspension DU4475 cells were directly harvested from the culture flask. For both cell lines, cells were fixed with a 4% para-formaldehyde (PFA, Sigma, Darmstadt, Germany) solution before being incubated in a 1 μM fluorescent conjugate PBS solution for 1 h. Three successive washes of 20 minutes were then conducted in PBS before imaging with a Cytation™3 MV (BioTek^®, Winooski, VT, USA) fluorescent microscopy module (M = 40×, fluorescence filters = GFP, RFP or Cy5). Cells mean fluorescence intensity was calculated with Gen5 3.08 software (BioTek^®) for three independent experiments. For each condition, fluorescence intensity was measured from 10 different imaging fields. For P-gp silencing, SUM1315 cells were exposed 72 hours to specific ABCB1 siRNAs diluted at 50 nM in culture medium and lipofectamine solution (Ambion®, reference AM51331 assay ID #4123/ABCB1, sequences 5′ > 3′: GGAUAUUAGGACCAUAAAUtt (sense), AUUUAUGGUCCUAAUAUCCtg (Antisense)). Cells were then fixed with para-formaldehyde 4% solution and stained with fluorescent conjugates or specific antibodies as previously described.

Daumar P., Goisnard A., Dubois C., Roux M., Depresle M., Penault-Llorca F., Bamdad M, & Mounetou E. (2023). Chemical biology fluorescent tools for in vitro investigation of the multidrug resistant P-glycoprotein (P-gp) expression in tumor cells. RSC Advances, 13(39), 27016-27035.

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Time-lapse Imaging of A549 Cells

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Brightfield and fluorescence (mCherry and mVenus) images of A549 WT and ERK-Fra1 cells were obtained using the Cytation5 cell imaging multi-mode reader (Biotek, Winooski, VT, USA). The 96-well plates were maintained at 37 °C with 5% CO₂ in a BioSpa 8 automated incubator (Biotek). Images were acquired over 12 h using a 20x objective and the Texas Red (586 nm excitation and 647 nm emission) and YFP (500 nm excitation and 542 nm emission) filter cubes (Biotek). Exposure, brightness, and contrast settings were constant across time points for each strain using the Gen5 3.08 software (Biotek).

Phillips S.M., Bergstrom C., Walker B., Wang G., Alfaro T., Stromberg Z.R, & Hess B.M. (2022). Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria. Pathogens, 11(2), 209.

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Anticancer Drug Screening on MDA-MB-231 Spheroids

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MDA-MB-231 spheroids cultured in OptiPASS^® medium without the storage step or stored for 3, 5 or 7 days at 4 °C in oxygen-free conditions were treated for 5 days (between the 5th and the 10th culture days), with increased concentrations of selected anticancer drugs. The size of the untreated control and treated spheroids was monitored daily using Cytation™5MV (Biotek^®, Winooski, VT, USA) coupled with BioSpa™ automated cell incubator (Biotek^®, Winooski, VT, USA) and Gen5 3.08 software (Biotek^®, Winooski, VT, USA). Growth curve slopes were calculated for each spheroid between the 5th and the 10th culture days. At the end of the treatment, spheroid viability was assessed by the Live/Dead^® and resazurin tests as described previously. Normalized viability was determined by the percentage of treated spheroids resorufin values on untreated control values.

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