Serum and saliva samples were thawed on ice, vortexed, and spun down at 16,000× g for 5 min at 4 °C. The samples were diluted (serum 1:1 and saliva 2:1) with the RD1-W buffer (R&D Systems, Abington, UK). All samples were analyzed with the custom-made 12-plex Luminex Mouse Discovery Assay kit (http://www.biotechne.com/g8AnddcM (accessed on 31 March 2022)) including CCL3/MIP-1α, KC, IP-10, G-CSF, IFN-γ, IL-1α, IL-1β, IL-6, IL-12 p70, MMP-9, TIMP-1, and TNF (Bio-Techne Ltd., Abington, UK). The plates with saliva samples were incubated overnight. A Luminex IS 200 instrument (Bio-Rad, Hercules, CA, USA) was used to record data. IL-1β, IL-6, IP-10, or IFN-γ were below the level of detection in saliva samples, while MMP-9 was excluded for both serum and saliva samples as the data were above the standard curve. Due to high background levels, IL-12 p70 was also excluded from the cytokine panel. Total protein concentrations in saliva samples were measured in mg/mL using spectrophotometry (Absorbance 280 nm, NanoDrop 2000c, Thermo Fisher Scientific, Waltham, MA, USA). The salivary cytokine levels were adjusted to total protein concentration and presented as (pg of cytokine)/(mg of total protein).
Luminex is 200 instrument
Manufactured by Bio-Rad
Sourced in United States
The Luminex IS 200 instrument is a multiplex platform that uses xMAP technology to detect and quantify multiple analytes simultaneously in a single sample. The instrument utilizes fluorescent-coded microspheres, known as Luminex Magnetic Microspheres, to capture and detect specific target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by Bio-Techne (1 mentions)
Il 1β, by Bio-Techne (1 mentions)
Mmp 9, by Bio-Techne (1 mentions)
Il 12p70, by Bio-Techne (1 mentions)
Tumor necrosis factor (tnf), by Bio-Techne (1 mentions)
Human cytokine 27 plex kit, by Bio-Rad (1 mentions)
Nine plex kit, by Bio-Rad (1 mentions)
Gibco cat no 10010 015, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bio plex pro wash station, by Bio-Rad (1 mentions)
3 protocols using luminex is 200 instrument
1
Multiplexed Cytokine Profiling of Serum and Saliva
2
Multiplex Serum Cytokine Analysis
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Serum cytokine levels were analyzed with a custom-made nine-plex kit (Cat No. 12014058, Bio-Rad Laboratories, Hercules, CA) containing eotaxin-1, granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-7, IL-8, interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. The nine cytokines were selected based on a screening of a representative set of samples with the human cytokine 27 plex kit (Cat No. M500KCAF0Y, Bio-Rad Laboratories). A 10 % bovine serum albumin (Cat No. A5403-50 G, lot SLBL9495 V, Sigma Aldrich, St. Louis, MO) solution in PBS (pH 7.4, Gibco Cat No. 10010-015, lot 2062123, Thermo Fisher Scientific, Waltham, MA) was added to all serum samples to a final concentration of 0.5% and vortexed. Then, samples were centrifuged for 10 min at 10,000×g at 4°C and 50μL of the supernatant was loaded onto the assay plate. Cytokine levels were measured on a Luminex IS 200 instrument (Bio-Rad). An in-house control was used to observe both intra and inter percentage coefficients of variation. Cytokine concentrations outside the reference limits that were extrapolated by the analysis software were also included in the statistical analysis.
3
Multiplex Cytokine Profiling in Plasma
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A custom 7 plex (cat. no.: 17007417, Bio-Rad Laboratories, Inc.) was used to determine plasma levels of IL-1 β, IL-1RA, IL-2, IL-2RA, IL-6, MIF, and TNF- α in patients and healthy volunteer samples. Samples were thawed, vortexed and kept on ice until centrifugation at 10 000xg/10min/4°C. The supernatant was further diluted (1+3) in separate tubes using the recommended sample diluent reagent and vortexed. Fifty microliters of the sample was loaded onto assay plate, whereas all samples were analysed in duplicate. The assay was optimized for “low level detection” by adding a standard point and extending the incubation of multi-plex beads and sample for up to one hour. All washing steps were performed with an automated magnetic plate washer (Bio-plex Pro wash station, Bio-Rad Laboratories, Inc. Hercules, CA, USA). The cytokine levels were determined using a Luminex IS200 instrument (Bio-Rad Laboratories, Inc. Hercules, CA, USA). A spiked plasma sample was used as a control to determine the percentage coefficient of variation (%CV). Intra %CV ranged from 0.5–10 and plate to plate variation (inter %CV) ranged from 5–19.
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