Aliquots of CVLs from various study visits in Segments A and B (see Fig 2 for exact time points) were assayed by Luminex (Bio-Plex MAGPIX, Bio-Rad Laboratories, Hercules, California, USA) for cytokines that have been associated with HIV transmission [33 (link)–38 (link)], utilizing a custom Invitrogen ProcartaPlex multiplex kit (ThermoFisher, Waltham, Massachusetts, USA) per manufacturer’s protocol. The analytes included: TNF-1α, IL-6, IL-1α, IL-1β, IL-1RA, MIP-1α, MIP-1β, RANTES, SDF-1α, IL-8, MCP-1, IP-10, GM-CSF, IFN-γ, IL-10, and IL-12p40.
Procartaplex multiplex kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ProcartaPlex multiplex kit is a product developed by Thermo Fisher Scientific for the simultaneous measurement of multiple analytes in a single sample. It enables the quantitative detection of various biomolecules, such as proteins, cytokines, or other targets, in a high-throughput and efficient manner.
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Lab products found in correlation
5 protocols using procartaplex multiplex kit
1
Cytokine Profile in HIV Transmission
2
Cytokine and Mucin Profiling in Murine Lungs
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Mice were anesthetized with isoflurane followed by overdose with ketamine. After tracheal cannulation, bronchoalveolar lavage (BAL) was performed with two 250 μL aliquots of PBS and subsequently combined together. The total number of cells in the BAL were counted using a Cytosmart automated cell counter (Corning). Total protein in the cell-free BAL was measured with the Pierce BCA Assay (Thermo Fisher Scientific). Inflammatory cytokines in the BAL were measured using a Luminex assay with a ProcartaPlex multiplex kit (Thermo Fisher Scientific) for the following analytes: eotaxin, granulocyte-macrophage colony stimulating factor (GM-CSF), CXC motif chemokine ligand 1 (CXCL1), interferon (IFN) -γ, interleukin (IL) -1β, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-2, IL-22, IL-23, IL-27, IL-4, IL-5, IL-6, IL-9, IFN-inducible protein (IP) -10, monocyte chemoattractant protein (MCP) -1, MCP-3, macrophage inflammatory protein (MIP) -1α, MIP-1β, MIP-2, regulated upon activation, normal T-cell expressed and secreted (RANTES), and tumor necrosis factor α (TNFα). Mucin 5 subtype AC (MUC5AC) in the BAL was measured by ELISA (Novus Biologicals).
3
Neuroinflammation Assay with BV2 Microglial Cells
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For neuroinflammation assays, BV2 microglial cells were pre-treated with compounds at non-toxic concentrations for 1 h and activated with 500 ng/mL LPS for 24 h. All the experiments were carried out as previously described 19 .
The levels of IL-6, TNF-α and IL-10 were evaluated with a ProCartaPlex Multiplex kit (Thermo Fisher Scientific), following manufacturer's instructions. Luminex 200 TM instrument and xPONENT ® software (LuminexCorp, Austin, TX) were used to collect the data.
The levels of NO were determined with Griess Reagent Kit, which evaluates the spontaneous oxidation of NO to nitrite under physiological conditions. Cells were cultured in DMEM without phenol red at 1x10 6 cells per well in 12-well plates and treated as described before. Then, 150 µL of medium were mixed with 20 µL of Griess reagent and 130 µL of deionized water. Samples were incubated during 30 min at room temperature and the absorbance was measured at 548 nm in a plate reader. All the experiments were performed three times by duplicate.
The levels of IL-6, TNF-α and IL-10 were evaluated with a ProCartaPlex Multiplex kit (Thermo Fisher Scientific), following manufacturer's instructions. Luminex 200 TM instrument and xPONENT ® software (LuminexCorp, Austin, TX) were used to collect the data.
The levels of NO were determined with Griess Reagent Kit, which evaluates the spontaneous oxidation of NO to nitrite under physiological conditions. Cells were cultured in DMEM without phenol red at 1x10 6 cells per well in 12-well plates and treated as described before. Then, 150 µL of medium were mixed with 20 µL of Griess reagent and 130 µL of deionized water. Samples were incubated during 30 min at room temperature and the absorbance was measured at 548 nm in a plate reader. All the experiments were performed three times by duplicate.
4
Cytokine Profiling of Stimulated DCs
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1×105 enriched blood DCs or sorted cord blood cDCs were stimulated in 200 µL DC medium/well in a 96-well U bottom plate at 37°C in a CO2 incubator for 18 hours. Stimulated cells were centrifuged at 300 × g for 5 min. DC supernatants were collected and cytokine concentrations were determined using either Cisbio human cytokine HTRF kits (Cisbio, #62HIL12PEG) or customized ProcartaPlex Multiplex kits (Life Technologies).
5
Naive T Cell Activation by cDCs
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1×105 sorted cord blood cDCs were stimulated in 200 µL DC medium/well in a 96-well U bottom plate for 18 hours at 37°C in a CO2 incubator. Stimulated cDCs were then washed three times with DC medium to remove remaining stimuli. Naive T cells were isolated from PBMCs using the Miltenyi Naive Pan T Cell Isolation Kit (#130-097-095) according to the manufacturer’s protocol. 1×105 isolated naive T cells were co-cultured 5:1 with 2×104 stimulated cDCs for 4 days at 37°C in a CO2 incubator. Cytokine in cell culture supernatants was quantified using the Cisbio human IFN-γ HTRF kit (#62HIFNGPET) or customized ProcartaPlex Multiplex kits (Life Technologies).
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