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15 protocols using mouse erythropoietin quantikine elisa kit

1

Hematological and Bone Marker Analysis

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Blood samples were collected by retro-orbital puncture under anesthesia. Hematological parameters were measured on an Advia 120 Hematology System (Bayer, Tarrytown, NY). Serum levels of erythropoietin were detected by ELISA according to the provided protocols (Human Erythropoietin Quantikine IVD ELISA kit, Mouse Erythropoietin Quantikine ELISA kit, R&D Systems). Serum levels of bone-related degradation products from C-terminal telopeptides of type I collagen (CTX-I) were detected by EIA according to the provided protocols (RatLaps (CTX-I), IDS).
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2

Measuring Plasma Erythropoietin and Hematocrit

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At each indicated time point in Figure 4, 27 μL of blood sample was collected from tail vein of each mouse and mixed immediately with 3 μL of 0.2M EDTA. The blood samples were then drawn into heparin-treated microhematocrit capillary tubes (Fisher Scientific) and centrifuged at 16,000 g for 3 min at 4oC using a micro-hematocrit centrifuge (Thermo Scientific). Hematocrit counts were then measured with a Critocaps micro-hematocrit tube reader (Leica Microsystems Inc). Then plasma samples in the capillary tubes were collected and stored at −80oC. Twenty-fold diluted plasma samples were then used for measuring plasma Epo protein concentration using Mouse Erythropoietin Quantikine ELISA Kit (R&D), and ELISA samples were measured with Victor X3 multi-label plate reader (PerkinElmer). Paired sample Student’s t-tests were performed to analyze the statistical significance of the plasma Epo concentration and hematocrit count differences among different treatment groups.
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3

Erythropoietin Quantification in Plasma

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Whole blood samples were centrifuged at 2000 × g for 10 min to pellet blood cells. The plasma was collected and stored at −70 °C until testing. Erythropoietin concentrations were measured by a mouse Erythropoietin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) as instructed.
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4

Erythropoietin Quantification by ELISA

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Cell culture supernatant were stored in a − 80 °C freezer after collection, transferred into a − 20 °C freezer 12–16 h prior to analysis and thawed on ice before analysis. The analysis was performed according to the protocol of provided in the Mouse Erythropoietin Quantikine ELISA Kit (R&D Systems).
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5

Measuring Plasma Erythropoietin and Hematocrit

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At each indicated time point in Figure 4, 27 μL of blood sample was collected from tail vein of each mouse and mixed immediately with 3 μL of 0.2M EDTA. The blood samples were then drawn into heparin-treated microhematocrit capillary tubes (Fisher Scientific) and centrifuged at 16,000 g for 3 min at 4oC using a micro-hematocrit centrifuge (Thermo Scientific). Hematocrit counts were then measured with a Critocaps micro-hematocrit tube reader (Leica Microsystems Inc). Then plasma samples in the capillary tubes were collected and stored at −80oC. Twenty-fold diluted plasma samples were then used for measuring plasma Epo protein concentration using Mouse Erythropoietin Quantikine ELISA Kit (R&D), and ELISA samples were measured with Victor X3 multi-label plate reader (PerkinElmer). Paired sample Student’s t-tests were performed to analyze the statistical significance of the plasma Epo concentration and hematocrit count differences among different treatment groups.
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6

Synthesis of Modified mRNA and Lipid Nanoparticles

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Cy5-labeled mRNA, ψ- 5’-triphosphate, m1ψ- 5’-triphosphate, m5C- 5’-triphosphate, s2U- 5’-triphosphate, and m5U- 5’-triphosphate were purchased from Trilink Biotechnologies (San Diego, CA). MEGAscript™ T7 Transcription Kit was purchased from Thermo Fisher Scientific (Waltham, MA). ScriptCap™ 2’-O-Methyltransferase Kit was purchased from CellScript (Madison, WI). Cholesterol and 1,2-epoxyhexadecane (C12) were purchased from Sigma Aldrich (St Louis, MO). The phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and C14-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL). DMG-PEG2000 was purchased from NOF America (White Plains, NY). The isodecyl acrylate (Oi10) amine and the 2[4-2(2-aminoethyl)amino)ethylpiperazine-1-YL)ethan-1-amine (200) were purchased from Sartomer (Colombes, France) and Enamine (Princeton, NJ), respectively. XenoLight D-Luciferin Potassium Salt was purchased from PerkinElmer (Waltham, MA). The lipid cKK-E12 was generously donated by the Anderson Lab at the Massachusetts Institute of Technology (Cambridge, MA). The Mouse Erythropoietin Quantikine ELISA Kit was purchased from R&D Systems (Minneapolis, MN).
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7

Quantifying Plasma EPO Levels

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Mice were injected intraperitoneally with 80 ng of purified α-toxin. Peripheral blood was obtained 24 hours after the injection via the vena cava using heparinized syringe. A mouse erythropoietin Quantikine ELISA kit (R&D Systems) was used to measure plasma EPO levels. The procedures were performed in accordance with the manufacturer’s instructions.
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8

Plasma Erythropoietin Quantification

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The plasma EPO level was measured using the Mouse Erythropoietin Quantikine ELISA kit (#MEP00B; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. In brief, thawed plasma was diluted 2-fold by calibrator diluents and then incubated on an antibody-coated microplate with each volume of assay diluents for 2 h using an orbital shaker. Washed wells were treated with antiserum conjugate for 2 h and with a substrate mixture for 30 min. The optical density measured at 450 and 540 nm was analyzed with the standards curve using the 4-parameter logistic curve-fit.
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9

Evaluating EPO Neuroprotective Effects

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The bioactivity of EPO secreted from the EPO-overexpressing NIH/3T3 cell line was examined in a cell model of neurodegeneration, PC12-INT-EGFP cells. 3T3, 3T3-EGFP, and EPO-3T3-EGFP cells (1.6 × 106 cells) were seeded in 10 cm dishes and cultured for 24 h. Culture supernatants were collected by sterile syringes and filtered through hydrophilic polyethersulfone membranes with a pore size of 0.22 μm (Millipore) for subsequent functional assays. The concentration of secreted EPO in collected culture supernatants was also measured using the Mouse Erythropoietin Quantikine ELISA Kit (R&D Systems). After 6 days of NGF induction, PC12-INT-EGFP cells were supplemented with conditioned media (50% v/v), which combined half of the fresh medium for PC12 cells and half of the collected culture supernatants, as mentioned previously. Moreover, 10 IU/mL of recombinant human EPO (hrEPO; specific activity, 115,336 IU/mg P; Millipore) was applied as the positive control. PC12 cells treated with fresh medium alone were used as the vehicle group. A cell viability assay, 48 h live-cell imaging, and immunocytochemistry were performed on PC12-INT-EGFP cells for testing the bioactivity of the secreted EPO.
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10

Neonatal and Adult Mouse Erythropoietin Levels

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The serum levels of Epo in neonatal (7 days old) and adult (8 to 12 weeks old) mice were quantified according to the manufacturer’s instructions for the Mouse Erythropoietin Quantikine ELISA Kit (R&D Systems). The expression of Epo mRNA was determined in the liver and kidney by qRT-PCR (see Supplementary Material for details). The statistical significance of relative expression changes in target mRNA levels was analyzed using the REST© 2009 software [10 ].
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