Kc junior software

Manufactured by Agilent Technologies

Sourced in United States

KC Junior is a software application developed by Agilent Technologies for laboratory data analysis. It provides basic functionality for data processing and visualization.

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KC Junior® microplate reader software (5 citations) KC Junior software package (4 citations)

50 protocols using kc junior software

Serological Assay for Antibody Detection

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Blood obtained at euthanasia by severing the abdominal aorta was clotted and centrifuged at 400 xg to obtain sera which were frozen in aliquots at −80°C for subsequent analysis. A sonicate of PC organisms (10µg protein/ml) was coated onto 96-well plates and then plates blocked with 5% dry milk in HBSS containing 0.05% Tween 20 as previously described (21 (link), 22 (link)). After extensive washing, sera and BALF samples (first washes) were diluted and incubated on plates overnight. Plates were extensively washed and bound IgG, IgM, or IgA was detected using alkaline phosphatase-conjugated anti-mouse IgG, IgM, or IgA (Sigma) followed by incubation with p-nitrophenylphosphate at 1mg/ml in diethanolamine buffer. Optical density was read at 405nm using a plate reader equipped with KC Junior software (Bio-Tek Instruments, Inc., Winooski, VT).

Opata M.M., Hollifield M.L., Lund F.E., Randall T.D., Dunn R., Garvy B.A, & Feola D.J. (2015). B lymphocytes are required during the early priming of CD4+ T cells for clearance of Pneumocystis infection in mice. Journal of immunology (Baltimore, Md. : 1950), 195(2), 611-620.

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Quantification of Serum MOG-specific IgG

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Animals were sacrificed by cardiac puncture and blood samples were allowed to clot at RT. Serum was obtained by centrifugation of clotted blood at 15,000×g for 30 min and frozen at -20C until use. Total serum MOG-specific IgG was quantified by plating serial serum dilutions on 96 well plates pre-coated with 10 ug/ml rMOG in coating buffer (Sodium bicarbonate). Plates were blocked with 1% BSA in PBS and incubated with sera overnight at 4C. After washing, MOG-specific IgG retained by the plate-bound rMOG was detected with alkaline phosphatase-conjugated goat-anti mouse IgG F(ab')₂ (Jackson Immunoresearch) and SigmaFast p-Nitrophenyl phosphate tablets with SigmaFast Tris Buffer tablets in dH2O (Sigma). Absorbance was measured at 450 nm with a μQuant plate reader and KC Junior software (Biotek) was used for data analysis.

Parker Harp C.R., Archambault A.S., Sim J., Ferris S.T., Mikesell R.J., Koni P.A., Shimoda M., Linington C., Russell J.H, & Wu G.F. (2015). B cell antigen presentation is sufficient to drive neuro-inflammation in an animal model of multiple sclerosis. Journal of immunology (Baltimore, Md. : 1950), 194(11), 5077-5084.

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Proinflammatory Cytokine Quantification

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Proinflammatory cytokines, including IL-6, TNF-α, and CRP, were assayed using ELISA kits (R&D Systems, Minneapolis, MN) for all participants. Fasting serum samples were collected in serum separator tubes, clotted for 30 minutes, and stored at −80°C until use. All assays were performed according to the vendor’s instructions. The final absorbance of each sample of the mixture was measured and analyzed at 450 nm using an ELISA plate reader with Bio-Tek Power Wave Xs and Bio-Tek’s KC junior software (Winooski, VT). The standard range was considered as specified in the vendor’s instructions. A linear regression R-square value of at least 0.95 was considered a reliable standard curve.

Chen M.H., Liu Y.L., Kuo H.W., Tsai S.J., Hsu J.W., Huang K.L., Tu P.C, & Bai Y.M. (2021). Neurofilament Light Chain Is a Novel Biomarker for Major Depression and Related Executive Dysfunction. International Journal of Neuropsychopharmacology, 25(2), 99-105.

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Quantification of Gonococcal Antibodies

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Samples of vaginal wash and serum were collected from the mice at the indicated time points.^{17 , 26 (link)} Gonococcus-specific IgA, IgG, IgM, and IgG subclass antibodies IgG1, IgG2a, IgG2b, and IgG3 in vaginal washes and sera were measured by ELISA on plates coated with whole gonococci, using undiluted vaginal wash and 10-fold diluted serum as starting dilutions. ^{17 , 26 (link)} Total IgA, IgG, and IgM concentrations in secretions were assayed by ELISA on plates coated with anti-IgA, -IgG, or -IgM antibodies (Southern Biotech, Birmingham, AL). H5 mouse monoclonal antibody (specific for N. gonorrhoeae porin serovar PIB3) or affinity-purified mouse IgA, IgG, and IgM (Southern Biotech) were used to establish standard curves. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse IgA, IgG, IgM, IgG1, IgG2a, IgG2b, or IgG3 antibody (Southern Biotech) and p-nitrophenylphosphate substrate (Southern Biotech). Plates were read in a VersaMax microplate reader with SoftMax software (Molecular Devices, Sunnyvale, CA) or an EL_X800 Universal microplate reader with KC Junior software (Bio-Tek Instruments, Winooski, VT). Antibody data were expressed as relative (fold increase) to the antibody levels detected in control samples (from sham-immunized mice) assayed simultaneously.

Liu Y., Hammer L.A., Liu W., Hobbs M.M., Zielke R.A., Sikora A.E., Jerse A.E., Egilmez N.K, & Russell M.W. (2017). Experimental Vaccine Induces Th1-driven Immune Responses and Resistance to Neisseria gonorrhoeae Infection in a Murine Model. Mucosal immunology, 10(6), 1594-1608.

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Synergistic Effects of BYL719 and Cisplatin

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To assess drug efficacy, 2×10³ cells from each cell line were seeded in 96-well plates and treated with 0.05–10 μM of BYL719 (SelleckChem, Houston, TX) or 0–20 μM of cisplatin to determine IC₅₀. After determining the IC₅₀ and optimal concentrations, all five cell lines were subjected to treatments of 1 μM of BYL719 and escalating concentrations of cisplatin (0 to 20 μM) either separately or in combination for 48 hours and then cell proliferation was analyzed with Cell Proliferation Kit (MTT) (Roche Diagnostic GmbH, Mannheim, Germany) to determine the effect of combination therapy on PIK3CA and p53 mutants. Combination index was calculated using the method of constant ratio drug combination proposed by the Chou and Talalay (39 (link)) and the COMPUSYN software (www.combosyn.com), where CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. These results were obtained using a 96-well spectrophotometer with KC Junior Software (Bio-Tek Instruments, Winooski, VT).

García-Carracedo D., Cai Y., Qiu W., Saeki K., Friedman R.A., Lee A., Li Y., Goldberg E.M., Stratikopoulos E.E., Parsons R., Lu C., Efstratiadis A., Philipone E.M., Yoon A.J, & Su G.H. (2020). PIK3CA and p53 mutations promote 4NQO-initated head and neck tumor progression and metastasis in mice. Molecular cancer research : MCR, 18(6), 822-834.

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Cytokine Quantification in T Cell Supernatants

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Cell-free supernatants from T cell cultures were assayed for IFNγ, IL-4 and IL-17A content using ELISA kits from eBioscience or BD Biosciences, according to the manufacturer’s instructions. Absorbance at 450 nm with a wavelength correction for 570 nm was determined using an ELx800 UV universal microplate reader (Biotek Instruments, Inc., Winooski, VT, United States) and KCjunior software (version 1.17; Biotek Instruments, Inc.). SOFTmax^® PRO software (version 4.3; Molecule Devices, Corp., Sunnyvale, CA, United States) was used to determine cytokine concentrations from the absorbance readings.

Furlong S., Coombs M.R., Ghassemi-Rad J, & Hoskin D.W. (2018). Thy-1 (CD90) Signaling Preferentially Promotes RORγt Expression and a Th17 Response. Frontiers in Cell and Developmental Biology, 6, 158.

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MTT Assay for Microglial Cell Viability

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MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma–Aldrich, St. Louis, MO, USA), was used to evaluate mitochondrial activity as an indirect measure of cell metabolic activity, as previously described (Redondo-Castro et al., 2013 (link); Alé et al., 2015 (link)). Microglial cells were incubated with LPS, extract lysate or extract lysate with ibuprofen (Sigma–Aldrich, St. Louis, MO, USA). After 24 h incubation, 0.15 mg/ml of MTT was added and the cells were incubated for 3 h at 37°C. The formazan crystals were dissolved in 200 μl of dimethyl sulfoxide (DMSO; Sigma–Aldrich, St. Louis, MO, USA) and 150 μl were passed to 96-well plates. The optimal density was determined with a microculture plate reader (Bio-Tek Instruments Inc., Germany) at 570 and 620 nm (to counteract the noise of the plastic) and analyzed with KCjunior™ software (Bio-Tek Instruments Inc., Germany). Three biological replicates were done.

Hernández J., Francos-Quijorna I., Redondo-Castro E., López-Vales R, & Navarro X. (2021). Microglia Stimulation by Protein Extract of Injured Rat Spinal Cord. A Novel In vitro Model for Studying Activated Microglia. Frontiers in Molecular Neuroscience, 14, 582497.

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Western Blot Analysis of Coilin and Related Proteins

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Western blot was performed according to the protocols described by Krejčí et al. [50 (link)]. To analyze coilin levels by western blot, we used an antibody raised against coilin (#sc-32860, Santa Cruz, USA) at a dilution of 1 : 1000. Coilin levels were analyzed in nondifferentiated and differentiated mESCs as well as embryonic and adult brains. In addition, we examined fibrillarin, histone deacetylase 1 (HDAC1), pan-acetylated lysine, and α-tubulin levels using the following antibodies: fibrillarin (#ab5821, Abcam, UK), HDAC1 (#sc7872, Santa Cruz Biotechnology, Inc., USA), anti-pan-acetylated lysine (#ab21623, Abcam, UK), and α-tubulin (#LF-PA0146, Thermo Fisher Scientific Inc., branch in Czech Republic). The secondary antibody was a peroxidase-conjugated anti-rabbit IgG (#A-4914; Sigma, Munich, Germany) diluted 1 : 2000. Equal amounts of protein were loaded in each gel lane. Protein levels were normalized to the total protein levels measured with a μQuant spectrophotometer and the KCjunior software (BioTek Instruments, Inc., Winooski, VT, USA) or to total histone H3 levels (#ab1791, Abcam, UK).

Krejčí J., Legartová S, & Bártová E. (2017). Neural Differentiation in HDAC1-Depleted Cells Is Accompanied by Coilin Downregulation and the Accumulation of Cajal Bodies in Nucleoli. Stem Cells International, 2017, 1021240.

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Quantifying TNF-α in Saliva and Serum

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Salivary and serum concentrations of TNF-α were determined by enzyme immunoassay by using the Quantikine immunoassay test (R&D Systems, Minneapolis, MIN, USA; cat. no. DTA00D) according to the manufacturer’s instructions. To determine the concentrations of the samples, a calibration curve was prepared by using the standards provided in the kit. Absorbance readings were performed by using the Universal Microplate Spectrophotometer (µQUANT BIO-TEK Inc., Bio-Tek World Headquarters, California, USA) at a wavelength of 450 nm. The results were processed with KCJunior software (Bio-Tek, Vinooski, VT, USA). The sensitivity of the kit was 6.23 pg/mL. The intra- and interassay errors were 3% and 8.4%, respectively.

Waniczek D., Świętochowska E., Śnietura M., Kiczmer P., Lorenc Z, & Muc-Wierzgoń M. (2022). Salivary Concentrations of Chemerin, α-Defensin 1, and TNF-α as Potential Biomarkers in the Early Diagnosis of Colorectal Cancer. Metabolites, 12(8), 704.

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Quantifying Neuronal Death via LDH Assay

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Neuronal death was quantified by measuring the release of LDH as previously described [46 (link)] using a colorimetric assay kit (Takara Biosciences, Madison, WI). Cell supernatant samples were diluted 1:4 in fresh medium prior to assay to avoid obtaining absorbance readings beyond the linear threshold. Absorbance was read at 490 nm using a µQuant microplate spectrophotometer (BioTek) and KC Junior software (BioTek). To quantify approximate levels of neuronal death, a standard curve was created as previously described [46 (link)] by lysing known quantities of neurons with 2 % Triton X-100 and measuring LDH release.

Davis S.M., Collier L.A., Leonardo C.C., Seifert H.A., Ajmo CT J.r, & Pennypacker K.R. (2016). Leukemia Inhibitory Factor Protects Neurons from Ischemic Damage via Upregulation of Superoxide Dismutase 3. Molecular Neurobiology, 54(1), 608-622.

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