Striatal tissue samples, obtained and homogenized as previously described, were also analyzed by performing Luminex assays to quantify levels of chemokine/cytokine (chemokine (C-X3-C motif) ligand 1 (CX3CL1), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-17a, and interferon (IFN)-γ; Millipore #RECYTMAG-65K), and for cell signaling (Akt, CREB, Erk, JNK, p65, p38, p70s6k, STAT3, and STAT5A; Millipore #48-680MAG). Analyses were performed according to the manufacturer’s recommendations.
Recytmag 65k
Manufactured by Merck Group
Sourced in United States, Germany
RECYTMAG-65K is a lab equipment product from Merck Group. It is a centrifuge device designed for the separation and isolation of biological samples. The product specifications and technical details are not available for a concise and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Elr timp1 cl 2, by MyBioSource (2 mentions)
Mbs705029, by Merck Group (2 mentions)
Mbs722532, by MyBioSource (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Luminex 200 system, by DiaSorin (1 mentions)
Eglp 35k, by Merck Group (1 mentions)
Ezrl 83k, by Merck Group (1 mentions)
80 insrt e01, by ALPCO (1 mentions)
Luminex 200 equipment, by DiaSorin (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Magpix, by DiaSorin (1 mentions)
Q view, by Quansys Biosciences (1 mentions)
Luminex 200 plate reader, by DiaSorin (1 mentions)
Protease, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Nf κb p65 transcription factor assay kit, by Cayman Chemical (1 mentions)
13 protocols using recytmag 65k
1
Multiplex Analysis of Striatal Signaling
2
Profiling AAA Tissue Proteome
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AAA tissue protein was extracted using RIPA buffer with proteinase inhibitor (Sigma #MCL1). Protein quantification was done by Bradford assay. For each AAA tissue samples 25ug of protein was analyzed for Tissue Inhibitor of Metalloproteinases 1 (TIMP1)-specific ELISA (RayBiotech, ELR-TIMP1-CL-2), MMP-9 (MyBioSource, MBS722532), CD68 (MyBioSource, MBS705029) and Cytokine multiplex assay (Millipore, RECYTMAG-65K) using manufacturer instructions.
3
Quantifying Retinal Cytokine Profiles
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Vehicle- and DIDS-treated retinas were harvested 2 and 24 h after the mechanical lesion and homogenized in an EDTA-free Protease Inhibitor Cocktail (Roche, #11836170001). Homogenates were centrifuged for 20 min at 14,000 G at 4 °C. BCA method was used to determine protein concentration, and bovine serum albumin was used as the standard. The following steps were performed according to the supplier’s instruction using the rat cytokine/chemokine magnetic bead panel (Millipore, #RECYTMAG-65K), and the cytokines GM-CSF, IL-4, IL-1B, IL-2, IL-6, IL-10, IL-12p70, INFy, IL-17, IP-10, and TNFa were simultaneously quantified. Cytokines values were normalized by the total protein concentration of each sample. Th1 and Th2 cytokines were divided according to the literature. Th1/Th2 dominance was determined by the INFy/IL-4 ratio. The mean of Pearson’s correlation was obtained in the correlation matrix report of heatmap statistical analysis using NCSS software (NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss.).
4
Quantifying AAA Tissue Biomarkers
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AAA tissue protein was extracted using RIPA buffer with proteinase inhibitor (Sigma #MCL1). Protein quantification was done by Bradford assay. For each AAA tissue samples 25ug of protein was analyzed for Tissue Inhibitor of Metalloproteinases 1 (TIMP1)-specific ELISA (RayBiotech, ELR-TIMP1-CL-2), MMP-9 (MyBioSource, MBS722532), CD68 (MyBioSource, MBS705029) and Cytokine multiplex assay (Millipore, RECYTMAG-65 K) using manufacturer instructions.
5
Serum CRP and Cytokine Quantification
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Blood samples were collected by the tail vein sampling method. Serum C-reactive protein (CRP) levels were detected with a Rat CRP ELISA kit (Immunology Consultants Laboratory, Inc., OR, United States). The CRP test kit is a highly sensitive two-site ELISA for measuring CRP in rat biological samples. The assay for quantification of CRP in samples requires that each test sample be diluted before use. For the serum samples of the rats in our study, 1/12000 dilution was appropriate for detection. The dilution ratio was determined based on the data provided by the kit and the data of the preliminary experiment.
The cytokines TNF-α, IL-6, IL-10, IL-1β, IL-4, and IL-17α were measured using a commercially available MILLIPLEX MAP Kit (Rat Cytokine/Chemokine Magnetic Bead Panel, 96-Well Plate Assay, Cat. #RECYTMAG-65K, RECYMAG65K27PMX, RECYMAG-65PMX27BK; Millipore Corporation, MA, United States) according to the manufacturer’s protocol. In this study, 1/2 dilution of serum was appropriate for detection. A Luminex 200 System (Luminex Corporation, TX, United States) was used for quantification.
The cytokines TNF-α, IL-6, IL-10, IL-1β, IL-4, and IL-17α were measured using a commercially available MILLIPLEX MAP Kit (Rat Cytokine/Chemokine Magnetic Bead Panel, 96-Well Plate Assay, Cat. #RECYTMAG-65K, RECYMAG65K27PMX, RECYMAG-65PMX27BK; Millipore Corporation, MA, United States) according to the manufacturer’s protocol. In this study, 1/2 dilution of serum was appropriate for detection. A Luminex 200 System (Luminex Corporation, TX, United States) was used for quantification.
6
Multimodal Metabolic Assessment in Rats
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Blood glucose was measured using glucometer, unless reading exceed glucometer capabilities, in which case, plasma glucose was measured using a glucose analyzer (Analox GM9, Stourbridge, UK). Fed and fasted plasma insulin was measured via ELISA following manufacturer’s protocol (Cat. # 80-INSRT-E01, Alpco, Salem, NH, USA). Circulating leptin at 8 week, 16 week, and 24 week timepoints was measured using multiplex assay following manufacturer’s protocol (Cat. # RECYTMAG-65K, Millipore, Burlington, MA, USA). Vena cava leptin and portal glucagon-like peptide-1 (GLP-1) from control and OFS-treated rats was determined by ELISA following manufacturer’s protocol (Cat. #EGLP-35K, Millipore; Cat. # EZRL-83K, Millipore).
7
Spinal Cord Inflammatory Protein Levels
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Pro- and anti-inflammatory protein levels in the spinal cord or dorsal root ganglia (DRG) samples were measured after 2 days of CFA injection. First, the L3-L5 spinal cord segment or DRG ipsilateral to the CFA-injected hind paw was dissected and stored at -80°C until analysis. Samples were homogenized in PBS with a protease inhibitor and a multiplexed immunoassay based on Luminex xMAP technology was performed for measuring IL-1β, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, IL-18, IFN-γ, GM-CSF, and VEGF (#RECYTMAG-65K, Millipore, Darmstadt, Germany). Assay plates were run according to the manufacturer’s protocol using a Luminex 200 equipment (Luminex, Austin, United States). Data were analyzed using the Luminex xPONENT® software version 3.1. Total protein concentration for each sample was measured using the PierceTM BCA Protein Assay Kit (Thermo Fisher, MA, United States). Contents for each sample were displayed in pg/μL of mg of total protein.
8
Quantitative Protein Profiling in Brain, Placenta, and Serum
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The total protein in each brain and placenta was extracted with RIPA buffer (Nacalai Tesque, Kyoto, Japan) followed by homogenization, and protein concentrations were measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc. MA USA). Whole blood was sampled from dams via their tail vein, incubated on ice, and centrifuged to purify the serum. Amniotic fluid was sampled from the uterus using a 25 G needle. All samples were stored at −80 °C until used in a Multiplex assay, and 25 μl of total protein (300–800 μg/ml) was used for the assay. A multiplexed immunoassay based on Luminex xMAP technology was performed using Rat Cytokine/Chemokine Magnetic Bead Panels for measuring RANTES, eotaxin, IL-1β, IL-12p70, IFN-γ, MCP-1, leptin, and VEGF (#RECYTMAG-65K https://www.emdmillipore.com/US/en , Millipore, Darmstadt, Germany) and Rat Vascular Injury Magnetic Bead Panels for measuring vWF, sE-selectin, sICAM-1, and adiponectin (#RV2MAG-26K, Millipore). Assays were performed according to the manufacturer’s instructions. Absolute values for each sample were adjusted relative to the protein concentration.
9
Multiplex Cytokine and Neuropeptide Analysis
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Animal samples were evaluated using a rat cytokine/chemokine panel (RECYTMAG-65K, Merck Millipore, Burlington, MA, USA) for TNF-α, IL-1β, IL-4, IL-6, IL-10, IL-17A, IFNγ, and CX3CL1, in the sciatic nerve and DRG, and by the rat neuropeptide panel (RECYTMAG-83K, Merck Millipore) for SP and β-endorphin in the plasma, using a commercially available multiplex magnetic bead-based kit (Magpix®, Luminex Corp., Austin, TX, USA). Tests were performed according to the manufacturer’s recommendations.
10
Quantifying Rat Cytokine/Chemokine Levels
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The collected ACM was used to quantify TNF, IL-6, IL-1β, and IFNγ using the MILLIPLEX MAP RAT cytokine/chemokine magnetic bead panel RECYTMAG-65K (Merck), following the manufacturer’s instructions. Data acquisition and analysis were performed in the LUMINEX platform using the Q-View (Quansys Biosciences) Software.
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