Discovery workbench 4

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The Discovery Workbench 4.0 software is a comprehensive data analysis and visualization platform designed for scientific research. It provides tools for importing, processing, and analyzing data from various sources, enabling researchers to explore and interpret their experimental findings.

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28 protocols using discovery workbench 4

Cytokine profiling in huNSG mice

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Serum of huNSG mice was collected upon terminal cardiac puncture using BD Mictrotainer tubes and frozen at −20°C until use. Samples were assayed for cytokines using the Meso Scale Discovery (Meso Scale Diagnostics, Rockville, MD) U-PLEX (Biomarker Group 1 Human, K15067L) and V-PLEX (Proinflammatory Panel 1 Human Kit, K15049D) platform following the manufacturer’s recommendations. Plates were read on a Meso QuickPlex SQ120 and analyzed with Discovery Workbench 4.0.12, both from Meso Scale Discovery. Samples and calibrator dilutions for the standard curves were run in duplicates. Heatmaps were generated in R using the superheat package (Barter and Yu, 2018 (link)) after empirical percentile transformation of cytokine levels (heatmaply package; Galili et al., 2018 (link)).
Plasma from Kenyan children samples was isolated from whole blood and kept frozen until use. IL-18 concentration in plasma of Kenyan children was measured using Human IL-18 Simplex ProcartaPlex from eBiosciences (cat#EPX01A-10267-901, lot#144494103, ULOQ/LLOQ of 49500/12 pg/ml). The assay was performed following the manufacturer’s protocol and run on a Bio-Plex 200 (Biorad) platform using the Bioplex Manager Software (Biorad, version 6.1). The concentration of the samples was calculated by plotting the expected concentration of the standards against the MFI generated by each standard using a5PL algorithm.

Caduff N., McHugh D., Rieble L., Forconi C.S., Ong’echa J.M., Oluoch P.O., Raykova A., Murer A., Böni M., Zuppiger L., Schulz T.F., Blackbourn D.J., Chijioke O., Moormann A.M, & Münz C. (2021). KSHV infection drives poorly cytotoxic CD56-negative natural killer cell differentiation in vivo upon KSHV/EBV dual infection. Cell reports, 35(5), 109056.

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Cytokine profiling in huNSG mice

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Tenover F.C., Jones R.N., Swenson J.M., Zimmer B., McAllister S, & Jorgensen J.H. (1999). Methods for Improved Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci: Results of a Multicenter Study. Journal of Clinical Microbiology, 37(12), 4051-4058.

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Protein Extraction and Cytokine Quantification from Wound Tissues

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For recovery of proteins, tissues within the wounds produced with 6‐mm biopsy punches were excised together with surrounding tissues using 8‐mm biopsy punches and placed in homogenisation tubes containing 1 mL of protein lysis buffer and a protease inhibitor cocktail (1:50). The tissue samples were homogenised and pelleted by centrifugation at 10 000 rpm for 10 minutes at 4°C to obtain a clean supernatant. Protein concentration in each sample was determined using the Bradford assay as previously described.³⁶ A custom U‐PLEX biomarker assay was used to measure the concentrations of six cytokines—IL‐6, IL‐1β, IL‐12p70, IL‐17F, GM‐CSF, and VEGF‐A—and four chemokines—CCL3, CXCL1, CXCL10, and CCL20 within the protein samples according to manufacturer's instructions (Meso Scale Discovery, Rockville, Maryland). Plates were analysed using a MESO QuickPlex SQ 120 (Meso Scale Discovery) that measures electrochemiluminescence. Concentrations of analytes were calculated via standard curves obtained using Discovery Workbench 4.012 software (Meso Scale Discovery).

Bounds K., Colmer‐Hamood J.A., Myntti M., Jeter R.M, & Hamood A.N. (2021). The influence of a biofilm‐dispersing wound gel on the wound healing process. International Wound Journal, 19(3), 553-572.

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Quantifying Rat Immunoglobulins and Inflammatory Markers

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Uncoated rat IgG (Catalog Number 88‐50490‐22, Invitrogen) and IgM (Catalog Number 88‐50540‐22, Invitrogen) ELISA kits were used to quantify IgG and IgM concentrations according to manufacturer instructions, using plasma that had been stored at −80 °C. Concentrations were determined from absorbance data by parametric interpolation using GraphPad Prism 8 software. Assessment of IL‐1β, IL‐6, TNF‐α and KC/GRO was performed using VPLEX Proinflammatory Rat Panel 2 kits (Catalog Number K15059D‐2, Meso Scale Diagnostics, Rockville, MD, USA) according to manufacturer instructions and was analysed using Discovery Workbench 4.0 (Meso Scale Diagnostics). All assays were performed in duplicate.

Williams K.M., Wang H., Paulsen M.J., Thakore A.D., Rieck M., Lucian H.J., Grady F., Hironaka C.E., Chien A.J., Farry J.M., Shin H.S., Jaatinen K.J., Eskandari A., Stapleton L.M., Steele A.N., Cohen J.E, & Woo Y.J. (2020). Safety of photosynthetic Synechococcus elongatus for in vivo cyanobacteria–mammalian symbiotic therapeutics. Microbial Biotechnology, 13(6), 1780-1792.

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Plasma Cytokine Profiling via ELISA

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Blood was collected in sodium heparin microcontainers. Plasma IL-1beta, IL-6, IL-8, and TNF-alpha were assessed using commercially available ELISA kits (V-PLEX Human Proinflammatory Panel II [4-Plex]) and a SECTOR Imager 2400A Model 1250. Samples were run in duplicate to ensure reproducibility. Analyses were done using DISCOVERY WORKBENCH 4.0 (Meso Scale Diagnostics (MSD), Rockville, MD).

Fazeli M.S., Pourrahmat M.M., Massah G., Lee K., Lavoie P.M., Fazeli M., Esser A, & Collet J.P. (2020). The Effect of Massage on the Cardiac Autonomic Nervous System and Markers of Inflammation in Night Shift Workers: a Pilot Randomized Crossover Trial. International Journal of Therapeutic Massage & Bodywork, 13(3), 6-17.

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Multiplex Immunoassay for Cytokine and Chemokine Profiling

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The V-PLEX Proinflammatory Panel 1 (human), Cytokine Panel 1 (human), and Chemokine Panel 1 (human) immunoassays (Meso Scale Discovery [MSD], Rockville, MD, USA) were used to measure the concentrations of cytokines/chemokines described in Table 1. Briefly, 50 μL of maternal plasma or calibrator were dispensed into the separate wells of the plates and incubated for 2 h with vigorous shaking at room temperature. The samples and calibrators were removed, and the plates were washed 3 times with the MSD 1X wash buffer, followed by the addition of 25 μL of the 1X detection antibody solution into each well. Plates were then incubated for 2 h with vigorous shaking at room temperature. The detection antibody was removed, and the plates were washed 3 times. Next, 150 μL of 2X Read Buffer T were added to each well, and the plates were read with the MESO QuickPlex SQ 120 (Meso Scale Discovery), and analyte concentrations were calculated with the Discovery Workbench 4.0 (Meso Scale Discovery). The assay characteristics are shown in Table 1.

Gallo D.M., Romero R., Bosco M., Chaiworapongsa T., Gomez-Lopez N., Arenas-Hernandez M., Jung E., Suksai M., Gotsch F., Erez O, & Tarca A.L. (2022). Maternal plasma cytokines and the subsequent risk of uterine atony and postpartum hemorrhage. Journal of Perinatal Medicine, 51(2), 219-232.

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Profiling Inflammatory Cytokines in Neonatal Whole Blood

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Blood was collected during the first and third postnatal weeks of life and stored as whole blood in a Microtainer™ with EDTA (BD, Franklin Lakes, NJ) at −20°C, as validated previously.^{22 (link),28 (link)} Calibration and blood samples were assayed in duplicate (n=475) using the MSD® V-Plex Custom Pro-inflammatory Panel 1 (Human) kit (Meso Scale Discovery®, Rockville, MD) to quantify levels of TNF-α, IL-6, IL-8, IFN-γ, IL-1β, and IL-10, and carried out according to manufacturer specifications. Samples were read using a SECTOR Imager 2400 and DISCOVERY WORKBENCH 4.0 (Meso Scale Discovery®).

Meister A.L., Gardner F.C., Browning K.N., Travagli R.A., Palmer C, & Doheny K.K. (2021). Vagal Tone and Pro-Inflammatory Cytokines Predict Feeding Intolerance and Necrotizing Enterocolitis-Risk. Advances in neonatal care : official journal of the National Association of Neonatal Nurses, 21(6), 452-461.

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Measuring IL-22 in Preterm Labor Amniotic Fluid

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Human amniotic fluid samples from women with spontaneous preterm labor were retrieved from the Biorepository of the Perinatology Research Branch (Table II). A U-PLEX immunoassay (Meso Scale Discovery, Rockville, MD) was used to measure the concentrations of IL-22 in the human amniotic fluid samples, according to the manufacturer’s instructions. The plate was read using the MESO QuickPlex SQ 120 (Meso Scale Discovery) and analyte concentrations were calculated with Discovery Workbench 4.0 (Meso Scale Discovery). The sensitivity of the IL-22 assay was 0.13 pg/mL.

Gershater M., Romero R., Arenas-Hernandez M., Galaz J., Motomura K., Tao L., Xu Y., Miller D., Pique-Regi R., Martinez G I.I.I., Liu Y., Jung E., Para R, & Gomez-Lopez N. (2022). Interleukin-22 Plays a Dual Role in the Amniotic Cavity: Tissue Injury and Host Defense against Microbes in Preterm Labor. Journal of immunology (Baltimore, Md. : 1950), 208(7), 1595-1615.

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Cytokine Profiling of Activated T Cells

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CD4⁺ T lymphocytes were isolated as described above, before being seeded at 1 x 10⁵ cells per well in 96 well plates. Cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for 48 h as described above. After 48 h the supernatants were collected and analysed using V-PLEX Pro-inflammatory Panel 1 (human) and V-PLEX Human IL-5 kits (Meso Scale Discovery) according to the manufacturer’s instructions. Data analysis was carried out using Discovery Workbench 4.0 (Meso Scale Discovery), before cytokine values were normalised to the total protein content of the well. Protein values were measured using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer’s instructions.

Miller J.R., Träger U., Andre R, & Tabrizi S.J. (2015). Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington’s Disease T Lymphocytes. PLoS ONE, 10(11), e0141793.

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Quantifying IL-6 in Serum and Placenta Explants

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IL-6 was examined using the U-PLEX Biomarker Group 1 Human Assays K15067L-1 immunoassay (Mesoscale Diagnostics, United States). All standards and serum and placenta explant supernatant samples were run in duplicate. Plates were prepared according to manufacturer’s instructions and analyzed on the Meso QuickPlex SQ 120. Results were generated as calculated concentration means on the Mesoscale (MSD) Discovery Workbench 4.0 assay analysis software. The MSD analysis software determines individual cytokine concentrations from electro-chemiluminescent signals via backfitting to the calibration curve. IL-6 concentration is presented in pg/mL.

Barron A., Manna S., McElwain C.J., Musumeci A., McCarthy F.P., O’Keeffe G.W, & McCarthy C.M. (2023). Maternal pre-eclampsia serum increases neurite growth and mitochondrial function through a potential IL-6-dependent mechanism in differentiated SH-SY5Y cells. Frontiers in Physiology, 13, 1043481.

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