Cytometric bead array (CBA) was performed using the mouse/rat soluble protein master buffer kit combined with the appropriate CBA flex sets (BD Biosciences), according to manufacturer’s instructions. Supernatants were diluted 2x in assay diluent before mixing with capture beads. Flow data were collected on a BD Fortessa and analyzed with FlowJo. Data represent the median fluorescence intensity of PE on beads collected for analysis, extrapolated to protein concentration using a standard curve.
Mouse rat soluble protein master buffer kit
Manufactured by BD
Sourced in United States
The Mouse/Rat Soluble Protein Master Buffer Kit is a laboratory equipment product designed to facilitate the extraction and preparation of soluble proteins from mouse and rat tissue samples. The kit provides the necessary buffers and reagents to efficiently extract and maintain the solubility of proteins from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cba flex set, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Cytometric bead array, by BD (2 mentions)
Facsdiva v9, by BD (2 mentions)
Mouse il 1α, by BD (2 mentions)
Mouse il 12 il 23p40, by BD (2 mentions)
Mouse il 10, by BD (2 mentions)
Mouse il 1β, by BD (2 mentions)
Mouse ifn γ, by BD (2 mentions)
Mouse il 6, by BD (2 mentions)
Mouse tnf α, by BD (2 mentions)
Fortessa, by BD (1 mentions)
Il 1β, by BD (1 mentions)
Ammonium chloride, by HiMedia (1 mentions)
5 protocols using mouse rat soluble protein master buffer kit
1
Quantifying Soluble Protein Levels
2
Mouse Hematopoietic Stem Cell Isolation
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Sodium chloride (S3014), potassium chloride (P9541), sodium phosphate dibasic (S3264), potassium phosphate monobasic (P9791), potassium bicarbonate (12602), ammonium chloride (A9434), ethylene diamine tetra acetic acid di-sodium salt (E6635), bovine serum albumin (BSA) (5482), absolute ethanol (100983), methanol (34860) were purchased from Sigma–Aldrich, St. Louis, MO, United States. CD 34, Sca 1, and CBA Flex Set which contains IL-3 (558346), IL-6 (558301), TNFα (558299), IFN γ (562233), G-CSF (560152), GM-CSF (558347), IL-1α (560157), IL-1β (560232), Mouse/Rat Soluble Protein Master Buffer Kit (558266) were procured from BD Biosciences, United States. May-Grunwalds Stain (S039) and Giemsa Stain Solution (TCL083) were procured from Hi-Media India.
1 L 1X PBS (8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium phosphate dibasic, 0.25 g of potassium phosphate monobasic to 1 L at pH 7.4), 1 L 1X RBC lysis buffer (1 g of potassium bicarbonate, 8 g of ammonium chloride and 0.03 g of di-sodium EDTA), 1% BSA in PBS, 70% ethanol in PBS, and may-grunwald-giemsa stain mixed in 3:1 ratio were prepared in the laboratory.
1 L 1X PBS (8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium phosphate dibasic, 0.25 g of potassium phosphate monobasic to 1 L at pH 7.4), 1 L 1X RBC lysis buffer (1 g of potassium bicarbonate, 8 g of ammonium chloride and 0.03 g of di-sodium EDTA), 1% BSA in PBS, 70% ethanol in PBS, and may-grunwald-giemsa stain mixed in 3:1 ratio were prepared in the laboratory.
3
Cytokine Profiling in Mice Serum
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Serum supernatant was isolated from the inferior vena cava of euthanized mice and cytokines measured using the Cytometric Bead Array (BD Biosciences) with the amount of capture beads, detection reagents, and sample volumes scaled down tenfold from manufacturer’s protocol. Data was collected on an LSRFortessa flow cytometer (BD Biosciences) with FACSDiva v9.0 (BD Biosciences) and analyzed with FlowJo v10 (BD Biosciences). Statistical outliers were removed in GraphPad Prism v9.3 using ROUT method (Q=1%). Cytokine used were mouse TNFα (BD 558299), mouse IL-6 (BD 558301), mouse IL-1α (BD 560157), mouse IL-1β (BD 560232), mouse IL-10 (BD 558300), mouse IL-12/IL-23p40 (BD 560151), and mouse IFN-γ (BD 558296) with Mouse/Rat Soluble Protein Master Buffer Kit (BD 558266).
4
Cytokine Profiling from Mouse Serum
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Serum supernatant was isolated from the inferior vena cava of euthanized mice, and cytokines measured using the Cytometric Bead Array (BD Biosciences), with the amount of capture beads, detection reagents, and sample volumes scaled down 10-fold from manufacturer’s protocol. Data were collected on an LSRFortessa flow cytometer (BD Biosciences) with FACSDiva v9.0 (BD Biosciences) and analyzed with FlowJo v10 (BD Biosciences). Statistical outliers were removed in GraphPad Prism v9.3 using ROUT method (Q = 1%). Cytokines used were mouse TNF-α (BD Biosciences 558299), mouse IL-6 (BD Biosciences 558301), mouse IL-1α (BD Biosciences 560157), mouse IL-1β (BD Biosciences 560232), mouse IL-10 (BD Biosciences 558300), mouse IL-12/IL-23p40 (BD Biosciences 560151), and mouse IFN-γ (BD Biosciences 558296) with Mouse/Rat Soluble Protein Master Buffer Kit (BD Biosciences 558266).
5
Quantifying Serum Cytokine Levels
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Serum cytokines interleukin-6 (IL-6), tumor necrosis factor (TNF), and interferon-γ (IFNγ) were quantified with reagents from BD Biosciences, including the Mouse/Rat Soluble Protein Master Buffer kit, according to the manufacturer’s instructions. In brief, serum samples were diluted 1:4 with the included assay diluent and mixed with capture beads specific for TNF and IL-6 and IFNγ. The PE detection reagent was added, and the samples were analyzed on a Fortessa Flow Cytometer. The resulting median fluorescence intensity (MFI) was used to calculate the cytokine levels in the serum as quantified in picograms per milliliter based on the standard curve. Values that did not fall into the range of the standard curve were entered as 0. The limit of detection for the IL-6, TNF, and IFNγ flex set kits was 1.4, 2.8, and 0.5 pg/mL, respectively.
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