Cytokine measurements in plasma and serum from each participant were obtained using the Human 62-multiplex array on the Luminex 200 IS system (Affymetrix) performed at the Stanford Human Immune Monitoring Center (HIMC). The manufacturer’s protocol was followed, with variations as described by Brodin et al.20 (link). Thawed plasma samples were centrifuged at maximal speed in a microcentrifuge for 10 min and supernatants entered in two replicate wells, ensuring all samples for each individual were in the same plate to avoid confounding sample processing condition with plate artifacts. Median fluorescence intensity (MFI) was preprocessed for each cytokine through a sequence of averaging over duplicate wells, natural-logarithm transformation to reduce variance heterogeneity, and isolation and removal of plate effects as previously reported21 (link). Average log2(MFI) per condition for each cytokine was calculated, and percent change from baseline for each subject was calculated and displayed in heatmaps. Cohort variance for each processing condition was also calculated and plotted into heatmaps.
Luminex 200 is system
Manufactured by Thermo Fisher Scientific
The Luminex 200 IS system is a multiplex assay platform that utilizes flow cytometry technology to perform quantitative and qualitative analysis of multiple analytes in a single sample. The system can simultaneously measure up to 100 different target molecules, including proteins, nucleic acids, and small molecules. The core function of the Luminex 200 IS system is to provide researchers and clinicians with a high-throughput, sensitive, and efficient method for multi-analyte detection and quantification.
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5 protocols using luminex 200 is system
1
Multiplex Cytokine Profiling in Plasma
2
Cytokine Secretion Profiling of PBMCs
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The secretion levels of cytokines or chemokines from PBMCs in supernatants were measured using a 62-multiplex assay on the Luminex 200 IS system (Affymetrix) performed by Stanford Human Immune Monitoring Center (HIMC). All samples were tested in duplicate wells. Data were analyzed using MasterPlex software (Hitachi Software Engineering America Ltd., MiraiBio Group), and the average of 2 median fluorescence intensity (MFI) values for each sample for each analyte were reported by Stanford HIMC. Then, the ratios were calculated by dividing the average MFI of each analyte for each sample by the average MFI of each analyte for complete RPMI medium control. These ratios were used to present the secretion level of each cytokine or chemokine from PBMCs for each sample.
3
Cytokine Secretion in Peanut Allergy
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The supernatants were harvested from the 3-day culture of mock-depleted PBMCs, CD3+ T cell–depleted PBMCs, or CD56+ cell–depleted PBMCs in the presence or absence of peanut protein. The secretion level of cytokines from PBMCs in supernatants were measured using the Luminex 200 IS system (Affymetrix) by Stanford’s Human Immune Monitoring Center (HIMC) and the MFI value for each analyte per sample was reported by Stanford’s HIMC.
4
Multiplex Cytokine Quantification from Plasma and Tissue
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Plasma was obtained after a 10-minute centrifugation (800g) of
fasting venous blood in heparin-sulfate collection tubes. For tissue protein
extracts, flash-frozen endoscopy biopsy samples stored at −80 °C
were thawed in NP-40 lysis buffer (Invitrogen) supplemented with protease and
phosphatase inhibitors (cOmplete-Mini, Roche; Halt, Thermo), homogenized on ice
(Tissue Master 125, Omni), centrifuged at 1000g, and
supernatants aliquoted and frozen until further use. Bicinchoninic acid (BCA)
protein quantification was performed (Thermo). Cytokine measurements in plasma
and tissue protein extracts were obtained using the Human 62-multiplex array on
the Luminex 200 IS system (Affymetrix) performed at the Stanford Human Immune
Monitoring Core as previously described (34 (link)). Briefly, equal protein concentration of samples was tested in
duplicate wells, and matched sets of cases and controls were always mixed in all
plates to reduce confounding case status with plate artifacts. Few samples
suffered from selective cytokine bead aggregation, leading to exclusion of these
cytokine/sample pairs from the analysis. Median fluorescence intensity data were
preprocessed for each cytokine through a sequence of averaging over duplicate
wells, natural-logarithm transformation to reduce variance heterogeneity, and
isolation and removal of plate effects as previously reported (34 (link)).
fasting venous blood in heparin-sulfate collection tubes. For tissue protein
extracts, flash-frozen endoscopy biopsy samples stored at −80 °C
were thawed in NP-40 lysis buffer (Invitrogen) supplemented with protease and
phosphatase inhibitors (cOmplete-Mini, Roche; Halt, Thermo), homogenized on ice
(Tissue Master 125, Omni), centrifuged at 1000g, and
supernatants aliquoted and frozen until further use. Bicinchoninic acid (BCA)
protein quantification was performed (Thermo). Cytokine measurements in plasma
and tissue protein extracts were obtained using the Human 62-multiplex array on
the Luminex 200 IS system (Affymetrix) performed at the Stanford Human Immune
Monitoring Core as previously described (34 (link)). Briefly, equal protein concentration of samples was tested in
duplicate wells, and matched sets of cases and controls were always mixed in all
plates to reduce confounding case status with plate artifacts. Few samples
suffered from selective cytokine bead aggregation, leading to exclusion of these
cytokine/sample pairs from the analysis. Median fluorescence intensity data were
preprocessed for each cytokine through a sequence of averaging over duplicate
wells, natural-logarithm transformation to reduce variance heterogeneity, and
isolation and removal of plate effects as previously reported (34 (link)).
5
Cytokine Profiling of Twin Peanut Allergy
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Cryopreserved PBMCs from six pairs of twins discordant for peanut allergy were thawed. For each condition of this experiment, PBMCs were cultured at 5 × 105 cells per 200 µl. After 3-d culture, the supernatants were harvested and stored at −80°C. The secretion level of cytokines or chemokines from PBMCs in supernatants were measured using 62-multiplex assay on the Luminex 200 IS system (Affymetrix) by the Stanford Human Immune Monitoring Center. All samples were tested in duplicate wells. Data were analyzed using MasterPlex software (Hitachi Software Engineering America; MiraiBio Group), and the average of two median fluorescence intensity (MFI) values for each sample for each analyte were reported by the Stanford Human Immune Monitoring Center. The ratios were calculated by dividing the average MFI of each analyte for each sample by the average MFI of each analyte for complete RPMI medium control. These ratios were used to present the secretion level of each cytokine or chemokine from PBMCs for each sample.
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