Victor x3 plate reader

Manufactured by PerkinElmer

Sourced in United States

The Victor X3 plate reader is a high-performance microplate reader designed for a variety of applications. It offers multi-mode detection capabilities, including absorbance, fluorescence, and luminescence measurements. The Victor X3 provides reliable and accurate data acquisition for researchers in life science, drug discovery, and other laboratory settings.

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Victor X3 (440 citations) Victor plate reader (111 citations) Victor X3 reader (16 citations)

94 protocols using victor x3 plate reader

Measuring Cell Proliferation in 2D and 3D

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To determine the proliferation rate in 2D or 3D, cells were cultured for ≤8 days in a 48-well plate as described in the “Dormancy assay” section. At the desired timepoints, 20 μL of PrestoBlue cell-viability reagent (Invitrogen) was added directly to the media and the plate was incubated for 10 min at 37 °C in the dark. Subsequently, fluorescence at wavelength 544/590 nm was captured using the VICTOR X3 plate reader (PerkinElmer). Measurements were normalized to controls with only Matrigel and/or media to adjust for background signals.
calcein-AM was additionally used to determine proliferation^{46 (link)}. In short, cells were washed twice with phosphate-buffed saline (PBS) after which 5 µM calcein-AM (Thermofisher, C1430) in PBS was added. After incubating for 30 min at 37 °C in the dark, fluorescence was captured at wavelength 494/517 nm using the VICTOR X3 plate reader (PerkinElmer). Afterward, measurements were normalized to controls with only Matrigel and/or media to adjust for background signals.

Prunier C., Alay A., van Dijk M., Ammerlaan K.L., van Gelderen S., Marvin D.L., Teunisse A., Slieker R.C., Szuhai K., Jochemsen A.G., Solé X., ten Dijke P, & Ritsma L. (2021). Breast cancer dormancy is associated with a 4NG1 state and not senescence. NPJ Breast Cancer, 7, 140.

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Screening and Characterization of TLR Agonists

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To screen and characterized TLR agonists, we utilized HEK293-derived HEK-Blue TLR reporter cell lines obtained from InvivoGen. Cells were cultured in accordance with manufacturer's instructions, and assays were optimized to ensure appropriate positive control performance. Briefly, cells were maintained in DMEM, 4.5 g/L glucose media supplemented with 2 mM glutagro, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Corning, NY), 10% heat-inactivated (HI) fetal bovine serum (FBS) (Sigma), 100 μg/mL Normocin (InvivoGen), and appropriate selective antibiotics. Compound dilutions were prepared in culture media and added to the culture plates prior to cell seeding. Cells were cultured with compounds for 16–24 h at 37°C in 5% CO₂. To measure production of SEAP, 20 μL of culture supernatant were transferred into 180 μL of Quanti-Blue detection medium (InvivoGen), incubated for 2–6 h at 37°C, and measured at optical density (OD) 630 nm using Victor X3 plate reader (PerkinElmer). Post-assay viability of HEK-Blue cells was determined following 4–6-h incubation with alamarBlue (Bio-Rad) by measuring fluorescence (560 nm excitation, 590 nm emission) on Victor X3 plate reader (PerkinElmer). EC50s of SEAP expression were calculated in Prism 8 (GraphPad) based on triplicates expressed as percentage of maximum activation by the positive control, typically 2 μM ODN 2006 (InvivoGen).

Ushach I., Zhu R., Rosler E., Pandey R.K., De Costa N.T., Pourshahian S., Han Q., Li C., Beigelman L., Gryaznov S.M, & Yun T. (2022). Targeting TLR9 agonists to secondary lymphoid organs induces potent immune responses against HBV infection. Molecular Therapy. Nucleic Acids, 27, 1103-1115.

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Quantitative Biofilm Analysis of Vibrio cholerae

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The quantitative analysis of biofilm formation was performed as reported previously^{13 (link)} with minor modifications. Overnight cultures of Vibrio cholerae were diluted 1:100 in LB media supplemented with either 1% DMSO or 100 μM (S)-sebastenoic acid. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 100 μM when indicated. 100 μL of the diluted culture were grown in polyvinyl chloride (PVC) 96 well plates with “U” bottom (Corning) for 10 hours at 30°C under static conditions. A negative control of cell-free LB media was analyzed under the same conditions. The grown cultures (planktonic cells) were transferred to clear 96 well flat bottom plates (Corning) and the Optical Density at 595 nm (OD₅₉₅) was measured using a Perkin Elmer Victor X3 plate reader. Each well was washed with sterile water to remove the remaining planktonic culture. 100 μL of 1% crystal violet (CV) was added to each well and incubated for 20 minutes at room temperature. CV was discarded and the wells were washed thoroughly with water. The wells were dried overnight and then 150 μL of 95% Ethanol were added. The CV adhered to the biofilms was solubilized by pipet mixing. 100 μL of each solution was transferred to clear 96 well flat bottom plates (Corning) and OD₅₉₅ values measured using a Perkin Elmer Victor X3 plate reader. Six independent biological replicates were analyzed.

Warner C.J., Salinas M., Zamorano-Sánchez D., Bray W.M., Lokey R.S., Yildiz F.H, & Linington R.G. (2017). The Bioactive Lipid (S)-Sebastenoic Acid Impacts Motility and Dispersion in Vibrio cholerae. Canadian journal of chemistry, 96(2), 196-203.

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TZM-bl Neutralization Assay Protocol

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TZM-bl neutralization assays were performed as previously described [15 (link),16 (link),43 (link)]. Briefly, eCD4-Ig or antibody titrations were incubated with infectious viruses for 1 hour at 37°C. TZM-bl cells were diluted in DMEM to 100,000 cells/mL and added to the virus/inhibitor mix. Cells were then incubated for 40 hours at 37°C. Viral entry was determined by luciferase readout with BriteLite Plus (Perkin Elmer, Waltham, MA) and read on a Victor X3 plate reader (Perkin Elmer, Waltham, MA).

Davis-Gardner M.E., Gardner M.R., Alfant B, & Farzan M. (2017). eCD4-Ig promotes ADCC activity of sera from HIV-1-infected patients. PLoS Pathogens, 13(12), e1006786.

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ADCC Assay for HIV Neutralization

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ADCC assay was performed as previously described [31 (link),32 (link)]. Briefly, CEM.NKR-CCR5-LTR-Luc target cells were infected by spinoculation with 89.6 (400ng p24) and NL4-3 (200ng p24) 4 days and YU-2 (500ng p24) 3 days prior to assay. Infection amounts for each assay were determined by virus titration on the target cells. On the day of the assay, infected target cells were mixed with NK effector cells at a 10:1 ratio in the presence of antibodies or eCD4-Ig. Cell and inhibitor mixes were incubated for 8 hours at 37°C. ADCC activity was measured by luciferase using BriteLite Plus (Perkin Elmer, Waltham, MA) and measured using a Victor X3 plate reader (Perkin Elmer, Waltham, MA).

Davis-Gardner M.E., Gardner M.R., Alfant B, & Farzan M. (2017). eCD4-Ig promotes ADCC activity of sera from HIV-1-infected patients. PLoS Pathogens, 13(12), e1006786.

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SARS-CoV-2 Neutralization Assay Protocol

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TZM-bl neutralization assays were performed as previously described (41 (link), 43 (link), 44 (link)). Briefly, antibody titrations were incubated with pseudotyped viruses for 1 h at 37°C. TZM-bl cells were diluted in DMEM to 100,000 cells/ml and added to the virus/inhibitor mix. Cells were then incubated for 48 h at 37°C. Viral entry was determined by luciferase readout with BriteLite Plus (Perkin Elmer, Waltham, MA) and read on a Victor X3 plate reader (Perkin Elmer, Waltham, MA).

Davis-Gardner M.E., Alfant B., Weber J.A., Gardner M.R, & Farzan M. (2020). A Bispecific Antibody That Simultaneously Recognizes the V2- and V3-Glycan Epitopes of the HIV-1 Envelope Glycoprotein Is Broader and More Potent than Its Parental Antibodies. mBio, 11(1), e03080-19.

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Cell Survival Assay for 6-TG Sensitivity

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A modified cell survival assay that measures DNA content over a 7-day period was employed (48 (link)). A stock concentration of 6-TG (Sigma, St. Louis, MO, USA) was prepared in 0.1 M NaOH and stored at −80°C. Cells (20 000 cells/well) were seeded into 48-well plates in 0.5 ml media on day 0. The next day (day 1), media were aspirated and 0.5 ml media containing indicated concentrations of 6-TG (μM) were added. Cells were exposed for 48 h, >2 doublings. Media was then removed, cells washed with PBS and fresh media were added. Cells were then allowed to grow for 7 days. Cells were then lysed in water, freeze-thawed and DNA content (a measure of cell growth) was determined by Hoescht 33258 fluorescence (Sigma, St. Louis, MO, USA) using a Victor X3 plate reader (PerkinElmer, Waltham, MA, USA). Data (means, ±SD) were expressed as treated/control (T/C) values from experiments performed at least three times in triplicate each. P-values were obtained using two-tailed Student's t-tests.

Patidar P.L., Motea E.A., Fattah F.J., Zhou Y., Morales J.C., Xie Y., Garner H.R, & Boothman D.A. (2016). The Kub5-Hera/RPRD1B interactome: a novel role in preserving genetic stability by regulating DNA mismatch repair. Nucleic Acids Research, 44(4), 1718-1731.

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Transcytosis Assay of Gluc-Fc Fusion Proteins

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The production and purification of Fc-Guassia luciferase (Gluc) fusion proteins (Gluc-Fc) was previously described (Nelms et al., 2017 ). For the transcytosis assays, cells were cultured on transwell filters (Corning) and then media was exchanged for Hank’s Balanced Salt Solution (HBSS), 20mM MES, and 1μg/ml Gluc-Fc, pH6.0 on the input side, and HBSS, pH7.4 on the output side. Filters were incubated for 90 minutes at 37°C, and then media of the output side was collected. To measure the concentration of Gluc-Fc, 80μL coelenterazine (Nano Light Technology) was added to 20μL media in a white 96-well plate and luciferase activity was measured using a Victor X3 plate reader (PerkinElmer).

Maeda K., Zachos N.C., Orzalli M.H., Schmieder S.S., Chang D., Gwilt K.B., Doucet M., Baetz N.W., Lee S., Crawford S.E., Estes M.K., Kagan J.C., Turner J.R, & Lencer W.I. (2022). Depletion of the apical endosome in response to viruses and bacterial toxins provides cell-autonomous host defense at mucosal surfaces. Cell host & microbe, 30(2), 216-231.e5.

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Measuring Apoptosis in MCM Absence

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To measure the rate of apoptosis in the absence of MCM, activities of apoptotic markers, caspase-3 and -7^{(22 (link))}, were measured using the caspase-Glo 3/7 assay (Promega, WI). RMH359 cells were plated at two different densities (400 cells and 4000 cells) in 96-well plates and allowed to attach for 24 hours in hybridoma growth medium supplemented with 50% MCM before switching to 100 μL of medium containing either 50% MCM or control medium and grown for an additional 7, 24, 48, and 72 hours. A 1:2 dilution of caspase 3/7 luminogenic substrate containing the caspase target sequence (DEVD) was added to each well and incubated for 1 hour. Cleavage of the DEVD sequence by caspase-3 and -7 results in a luminescent signal that is proportional to their cellular activity.^{(23 (link),24 )} Luminescence was measured on a Victor X3 plate reader for 0.2 seconds (PerkinElmer, MA) and data represent 12 replicates per time point and are expressed as mean counts per second (CPS) ± SEM.

Hnasko R., Lin A.V., Stanker L, & McGarvey J. (2018). A Bioassay for Optimization of Macrophage-Conditioned Medium as a Culture Supplement to Promote Hybridoma Cell Survival and Growth. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 37(3), 126-133.

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Pseudovirus-Based Neutralizing Antibody Assay

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The measurement of neutralizing antibodies elicited after vaccination was assessed as described elsewhere [13 (link)]. In brief, EBOV or MARV GP-specific neutralizing antibody was assessed by using a single-round infection assay with EBOV or MARV GP-pseudotyped lentiviruses, respectively, containing the luciferase reporter gene. The derivative T-Ag–expressing 293T cells were used as infection targets and incubated in a 96-well plate 1 day before infection with pseudovirus in the presence of a 1:100 dilution of subject serum samples. EBOV or MARV GP-pseudotyped lentiviral virions were produced as described elsewhere [16 (link)]. Pre- and postimmune serum samples were tested as indicated in the figure legends. Cells were lysed 72 hours after infection and assayed with the Luciferase Assay System (Promega, E1501/E1531), using a Victor X3 Plate Reader from PerkinElmer to detect luciferase activity.

Sarwar U.N., Costner P., Enama M.E., Berkowitz N., Hu Z., Hendel C.S., Sitar S., Plummer S., Mulangu S., Bailer R.T., Koup R.A., Mascola J.R., Nabel G.J., Sullivan N.J., Graham B.S, & Ledgerwood J.E. (2014). Safety and Immunogenicity of DNA Vaccines Encoding Ebolavirus and Marburgvirus Wild-Type Glycoproteins in a Phase I Clinical Trial. The Journal of Infectious Diseases, 211(4), 549-557.

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