Cytokine concentration in cell-free supernatants was determined for 2D monolayer cell and 3D spheroid models. At first, 5 × 103 monolayer target cells were incubated for 24 h with UniCAR T-cells in the absence or presence of αFAP TM at an E:T ratio of 5:1. For the 3D cell model the same setup was performed with the monolayer target cells being replaced by single spheroids. The concentration of each cytokine was determined using the MACSPlex Cytokine 12 Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Data acquisition and analysis were performed using a MACSQuant® Analyzer and the MACSQuantify® software (Miltenyi Biotec).
Macsplex cytokine 12 kit
Manufactured by Miltenyi Biotec
Sourced in Italy, United States, Germany
The MACSPlex Cytokine 12 Kit is a multiplex assay that allows for the simultaneous quantification of 12 different cytokines from a single sample. The kit utilizes magnetic bead-based technology to capture and detect the cytokines, providing a comprehensive analysis of the cytokine profile in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Macsquant analyzer, by Miltenyi Biotec (5 mentions)
Macsquantify software, by Miltenyi Biotec (4 mentions)
Macsquant analyzer 10 flow cytometer, by Miltenyi Biotec (2 mentions)
Human il 1 beta il 1f2 quantikine elisa kit, by R&D Systems (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Flowlogic software, by Inivai Technologies (1 mentions)
Opteia human tnf elisa kit, by BD (1 mentions)
Opteia human il 2, by BD (1 mentions)
Opteia human ifn γ, by BD (1 mentions)
Macsquantify, by Miltenyi Biotec (1 mentions)
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Macsquant analyzer 8, by Miltenyi Biotec (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Texmacs medium, by Miltenyi Biotec (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Macsquantify 2, by Miltenyi Biotec (1 mentions)
Opteia human elisa kit, by BD (1 mentions)
αcd69 apc, by Miltenyi Biotec (1 mentions)
21 protocols using macsplex cytokine 12 kit
1
Cytokine Profiling of 2D and 3D Cell Models
2
Cytokine Profiling of T-Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Detection of cytokines (GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A and TNF-α) was performed using the MACSPlex Cytokine 12 Kit from Miltenyi Biotec following the manufacturer’s protocol. Cell acquisition was performed at the MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec) using the Express Modes MACSPlex_Standard and MACSPlex_Sample. Calibration of the MACSQuant 10 was performed prior to measurement with MACSQuant Calibration Beads. Flow-cytometry results were analysed using MACSQuantify 2.13.1 software. Undiluted medium (harvested on day 9 after restimulation) from runs #1-#4 was used in duplicates for this assay. Medium from non-edited CD4+-T cells from small-scale experiments harvested on day 12 after first stimulation was used as a control in duplicates. Data represents average results of three different donors. The detailed protocol for small-scale cell cultivation and the obtained data are described in Schwarze et al [25 ].
3
Cytokine Profiling Before and After Ruxolitinib Therapy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Supernatants from monocytes cultures in the presence/absence of LPS stimulation (4/24 hours) and before/after 6 months of RUX therapy or with/without in vitro RUX treatment were harvested (centrifugation at 400 g) and frozen at −80°C with 1% of DMSO until assays were performed. Cytokine concentration was determined by commercially available MACSPlex Cytokine 12 Kit (Miltenyi Biotec, Bologna, Italy) for human IL6, TNF-α and IL10 according to the manufacturer’s instruction. IL1β was determined by Human IL1 beta/IL1F2 Quantikine ELISA Kit (R&D Systems, Milan, Italy).
4
Cytokine Profiling via MACSPlex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Supernatant was taken from each sample after incubation, and TNF‐α, IFN‐α and IL‐6 were measured with the MACSPlex cytokine 12 Kit (Miltenyi Biotech), according to the manufacturer's instructions.
5
Multiplex Serum Cytokine Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The serum concentration of IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, TNF-α, GM-CSF was measured using the MACSPlex Cytokine 12 kit (Miltenyi Biotec). Samples were prepared as recommended by the manufacturer. Briefly, samples were centrifuged at 10000g for 5 minutes at 4°C to remove large debris and the supernatant was then diluted 1:4 with sample buffer provided within the kit. Data were acquired on a CytoFlex flow cytometer (Beckman Coulter) at a 20 µL/min flow rate. Acquisition stopping rule was set at 4000 events in the bead gate or 180µl of acquired sample. Exported data were analysed with Flowlogic software (Inivai Technologies) and the median intensity in APC was used to extrapolate cytokine concentrations. To all samples having concentration values out of range (OOR), an arbitrary value corresponding to half of the lowest measured concentration was assigned.
6
Cytokine Profiling of Activated T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For activation experiments, 1×105 gene modified T cells were seeded in 96-well plates in triplets together with target cells. TMs were added at the indicated concentrations. After a 48h- cultivation CD25 surface expression on T cells was analyzed using a MACSQuant Analyzer®. Cell free supernatants were harvested after 24h from cultures to determine cytokine concentrations by using OptEIA™ Human IFN-γ, OptEIA™ Human IL-2, OptEIA™ Human IL-6, and OptEIA™ Human TNF ELISA Kits (BD Biosciences, Heidelberg, Germany). Alternatively, cell culture supernatants were analyzed using the MACSPlex Cytokine 12 Kit (Miltenyi Biotec GmbH), a MACSQuant® Analyzer (Miltenyi Biotec GmbH) and the MACSQuantify® software (Miltenyi Biotec GmbH) according to the manufacturer‘s instructions.
7
Cytokine Secretion Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the levels of secreted cytokines, cell-free supernatants from the co-culture of target cells and UniCAR T-cells in the presence or absence of TM were harvest after 24 h incubation. Cytokine concentrations were quantified using the MACSPlex Cytokine 12 Kit (Miltenyi Biotec) according to the manufacturer’s instructions.
8
Cytokine release analysis of UniCAR T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UniCAR T cells were cultivated with or without target cells at an e:t ratio of 5:1 either in the presence or absence of 30 nM anti-CD19 TM. After 24h, cell-free supernatants were harvested and analyzed using the MACSPlex Cytokine 12 Kit, human, a MACSQuant® Analyzer and the MACSQuantify® software (all from Miltenyi Biotec) according to the manufacturer‘s instructions [39 (link), 40 (link)].
9
Serum Cytokine Profiling Using MACS Plex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Normal serum and DS were subjected to cytokine analysis by using the MACS Plex Cytokine 12 kit (Miltenyi Biotec, NJ, USA) on fluorescence activated cell sorter as per the manufacturer's instructions. Serum samples were mixed with MACS Plex Cytokine Capture Beads and incubated for 2 h on a shaker in the dark. After incubation, the pellet of beads was obtained by centrifugation. The pellet was washed with 200 μL MACS Plex buffer twice followed by incubation with MACS Plex Cytokine 12 detection reagent for 1 h on a shaker in the dark. Samples were centrifuged, and the pellet was suspended in 200 μL MACS Plex buffer, which was acquired on Attune NxT acoustic focusing cytometer (Thermo Fisher). Results obtained from the cytometer were converted into pg/mL after correlating with standards. Cytokine levels were normalized to the mg of protein/well.
10
Cytokine Release and Cytolytic Activity of Anti-CD20 CAR T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 × 105 JeKo-1 and 1 × 105 CAR T cells were co-cultured in TexMACS™ Medium (Miltenyi Biotec) for 24 h in 96-well round bottom plates. Supernatants were collected at the endpoint and used to detect the cytokines released by anti-CD20 CAR T cells using the MACSPlex Cytokine 12 Kit (Miltenyi Biotec) with the four selected human cytokines IFN-γ, IL-2, TNF-α and GM-CSF, according to the manufacturer's instructions. The cytolytic activity of the engineered T cells was evaluated by using 1 × 104 CD20+ JeKo-1 cells labeled with 1 μM CellTraceTM Violet (Life Technologies), as target cells. Effector and target cells were co-cultured for 24 h at the indicated ratios (E:T) in 96-well round bottom plates. Detection of the specific lysis was performed by quantitation of Violet dye labeled target cells using a MACSQuant Analyzer 8 (Miltenyi Biotec). Mock-transduced T cells were used as control at the same effector-to-target ratios.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!