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Luminex flexmap 3d platform

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The Luminex Flexmap 3D platform is a multiplex assay system designed for quantitative and qualitative analysis of multiple analytes in a single sample. The core function of the platform is to enable simultaneous detection and measurement of up to 500 different analytes in a single well.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions) Protease inhibitor cocktail, by Merck Group (3 mentions) Milliplex map mouse cytokine chemokine magnetic bead panel, by Merck Group (3 mentions) Luminex kit, by Merck Group (2 mentions) Map mouse cytokine chemokine magnetic bead panel, by Merck Group (1 mentions) Bca assay kit, by Thermo Fisher Scientific (1 mentions) Bullet blender, by Next Advance (1 mentions) Bio plex pro, by Bio-Rad (1 mentions) Human pd 1 duoset elisa, by R&D Systems (1 mentions) Elisa max standard set, by BioLegend (1 mentions)

7 protocols using luminex flexmap 3d platform

1

Quantifying Cytokine Levels in Lung Infections

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Determination of lung cytokine levels was performed on homogenized whole lungs from infected animals harvested four- and 12- hours post-infection and homogenized in PBS. Samples were normalized to total protein prior to analysis. Determination of systemic cytokine levels was performed on serum isolated from infected mice at the indicated time points. Bone marrow-derived macrophage cytokines were quantified from cell culture supernatant at four hours post-infection. All cytokines were quantified using the MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore) on a Luminex Flexmap 3D platform (Luminex) according to the manufacturer’s recommended protocol.
Hood-Pishchany M.I., Pham L., Wijers C.D., Burns W.J., Boyd K.L., Palmer L.D., Skaar E.P, & Noto M.J. (2020). Broad-spectrum suppression of bacterial pneumonia by aminoglycoside-propagated Acinetobacter baumannii. PLoS Pathogens, 16(3), e1008374.
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2

Intranasal A. baumannii Infection in Mice

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Mice were infected intranasally with approximately 3 X 108 CFU of A. baumannii 17978 and CFU were enumerated as previously described (Palmer et al., 2019 (link)). Mice were euthanized at 8 h, 12 h, 24 h or 36 h post infection and lungs, livers, and spleens were removed aseptically. For the Luminex assay, lungs were harvested 8 h post infection, homogenized, and frozen at −80 prior to cytokine/chemokine analysis. Lung homogenates were thawed, and protease inhibitor cocktail (Sigma) was added at approximately 1.100 dilution. The protein concentration was determined by (BCA) assay using bovine serum albumin (BSA) as a standard and then normalized to 10 mg/mL prior to using the Luminex kit (Millipore) as instructed but using 1/3 bead concentration recommended by the manufacturer. Samples were run on the Luminex Flexmap 3D platform (Luminex, Austin TX).
Palmer L.D., Minor K.E., Mettlach J.A., Rivera E.S., Boyd K.L., Caprioli R.M., Spraggins J.M., Dalebroux Z.D, & Skaar E.P. (2020). Modulating Isoprenoid Biosynthesis Increases Lipooligosaccharides and Restores Acinetobacter baumannii Resistance to Host and Antibiotic Stress. Cell reports, 32(10), 108129.
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3

Cytokine Profiling in C. difficile Infection

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Cytokine analysis was performed on mice prior to infection (n=5/group) and 2 days following infection (n=5/group) with C. difficile strain R20291. At necropsy, whole ceca from mice fed altered Zn diets were flash frozen in liquid nitrogen and frozen at −80°C. Ceca were thawed, thoroughly homogenized in 1 mL of Pierce IP Lysis Buffer (Thermo Scientific), and pelleted at 4°C. Supernatants were collected and normalized to total protein content using a Pierce BCA Protein Assay Kit (Thermo Scientific). Cytokine levels were measured using the Milliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore) and the Luminex Flexmap 3D platform (Luminex).
Zackular J.P., Moore J.L., Jordan A.T., Juttukonda L.J., Noto M.J., Nicholson M.R., Crews J.D., Semler M.W., Zhang Y., Ware L.B., Washington M.K., Chazin W.J., Caprioli R.M, & Skaar E.P. (2016). Dietary Zinc Alters the Microbiota and Decreases Resistance to Clostridium difficile Infection. Nature medicine, 22(11), 1330-1334.
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4

Intranasal A. baumannii Infection in Mice

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Mice were infected intranasally with approximately 3 X 108 CFU of A. baumannii 17978 and CFU were enumerated as previously described (Palmer et al., 2019 (link)). Mice were euthanized at 8 h, 12 h, 24 h or 36 h post infection and lungs, livers, and spleens were removed aseptically. For the Luminex assay, lungs were harvested 8 h post infection, homogenized, and frozen at −80 prior to cytokine/chemokine analysis. Lung homogenates were thawed, and protease inhibitor cocktail (Sigma) was added at approximately 1.100 dilution. The protein concentration was determined by (BCA) assay using bovine serum albumin (BSA) as a standard and then normalized to 10 mg/mL prior to using the Luminex kit (Millipore) as instructed but using 1/3 bead concentration recommended by the manufacturer. Samples were run on the Luminex Flexmap 3D platform (Luminex, Austin TX).
Palmer L.D., Minor K.E., Mettlach J.A., Rivera E.S., Boyd K.L., Caprioli R.M., Spraggins J.M., Dalebroux Z.D, & Skaar E.P. (2020). Modulating Isoprenoid Biosynthesis Increases Lipooligosaccharides and Restores Acinetobacter baumannii Resistance to Host and Antibiotic Stress. Cell reports, 32(10), 108129.
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5

Cytokine Profiling of Infected Hearts

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Infected whole hearts were harvested 24 hours post infection, immediately frozen on dry ice, and stored at −80°C prior to analysis. Hearts were spiked with protease inhibitor cocktail (Sigma, St. Louis MO), thawed, and placed in Navy Bullet Blender tubes (Next Advance, Averill Park NY) in 200 μL PBS (Sigma). Hearts were homogenized at 4°C using setting 8 for 4 min and setting 10 for 2 min in the Bullet Blender (Next Advance, Averill Park NY). Beads and cells were pelleted at 4000 x g for 10 minutes at 4°C for 10 min and supernatants were collected. Protein concentrations in the supernatants was quantified by BCA assay kit (Thermo Fisher Scientific, Waltham, MA) and normalized to 10 mg/mL. Cytokines were assayed in supernatants using MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore, Darmstadt, Germany; catalog number: MCYTOMAG-70K) according to the manufacturer’s instructions. Samples were run on the Luminex Flexmap 3D platform (Luminex, Austin TX).
Juttukonda L.J., Berends E.T., Zackular J.P., Moore J.L., Stier M.T., Zhang Y., Schmitz J.E., Beavers W.N., Wijers C.D., Gilston B.A., Kehl-Fie T.E., Atkinson J., Washington M.K., Peebles R.S., Chazin W.J., Torres V.J., Caprioli R.M, & Skaar E.P. (2017). Dietary manganese promotes staphylococcal infection of the heart. Cell host & microbe, 22(4), 531-542.e8.
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6

Quantifying Cytokine and Immune Biomarkers

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CD4+ T cell culture supernatants were collected at day 5 and stored at −80°C. IFN‐γ levels were determined by enzyme‐linked immunosorbent assay (ELISA) using the ELISA MAX™ standard set (Biolegend). Levels of sPD‐1 were determined by human PD‐1 DuoSet ELISA (R&D Systems). Serum samples from HC donors and serum and paired cell‐free SF samples from patients with OA, RA or PsA were collected and stored at −80°C until analysed by ELISA (for sPD‐1; R&D Systems) or Bio‐Plex Pro™ (for TNFα, IL‐6 and IL‐1β; Bio‐Rad Laboratories) on the Luminex FlexMap 3D platform (Luminex Corporation, Austin, TX, USA). All assays were performed according to the manufacturers' protocols.
Bommarito D., Hall C., Taams L.S, & Corrigall V.M. (2017). Inflammatory cytokines compromise programmed cell death‐1 (PD‐1)‐mediated T cell suppression in inflammatory arthritis through up‐regulation of soluble PD‐1. Clinical and Experimental Immunology, 188(3), 455-466.
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7

Cytokine Profiling in C. difficile Infection

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Cytokine analysis was performed on mice prior to infection (n=5/group) and 2 days following infection (n=5/group) with C. difficile strain R20291. At necropsy, whole ceca from mice fed altered Zn diets were flash frozen in liquid nitrogen and frozen at −80°C. Ceca were thawed, thoroughly homogenized in 1 mL of Pierce IP Lysis Buffer (Thermo Scientific), and pelleted at 4°C. Supernatants were collected and normalized to total protein content using a Pierce BCA Protein Assay Kit (Thermo Scientific). Cytokine levels were measured using the Milliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore) and the Luminex Flexmap 3D platform (Luminex).
Zackular J.P., Moore J.L., Jordan A.T., Juttukonda L.J., Noto M.J., Nicholson M.R., Crews J.D., Semler M.W., Zhang Y., Ware L.B., Washington M.K., Chazin W.J., Caprioli R.M, & Skaar E.P. (2016). Dietary Zinc Alters the Microbiota and Decreases Resistance to Clostridium difficile Infection. Nature medicine, 22(11), 1330-1334.
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