Circulating concentrations of cell-free hemoglobin, haptoglobin and hemopexin were measured with commercially available solid-phase enzyme-linked immunosorbent assay kits (Hemoglobin Human ELISA Kit; ab157707, Abcam, Cambridge, UK; Haptoglobin ELISA Kit (Human) OKIA00064, Aviva Systems Biology, San Diego, CA, USA and Hemopexin ELISA Kit (Human) OKIA00066, Aviva Systems Biology, San Diego, CA, USA) according to the manufacturer’s instructions.
Human hemoglobin elisa kit
Manufactured by Abcam
Sourced in United Kingdom
The Human Hemoglobin ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human hemoglobin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Cell Signaling Technology (1 mentions)
Heparin sodium, by Merck Group (1 mentions)
Phospho enos ser1177, by Cell Signaling Technology (1 mentions)
Phospho p38 thr180 tyr182, by Cell Signaling Technology (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Vascular endothelial growth factor (vegf), by Merck Group (1 mentions)
Hochest 33258, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Vegf human elisa kit, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Cfda se cell proliferation assay and tracking kit, by Beyotime (1 mentions)
Ptk787, by Merck Group (1 mentions)
C raf, by Cell Signaling Technology (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Matrigel, by BD (1 mentions)
Luminescent atp detection assay kit, by Abcam (1 mentions)
U plex plus human alpha synuclein kit, by Mesoscale (1 mentions)
7 protocols using human hemoglobin elisa kit
1
Quantifying Circulating Hemoglobin Biomarkers
2
Characterization of Cellular Angiogenic Responses
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CA (purity>99.0%) was purchased from the Beijing Naturally Occurring Drugs Research Institute, China. A stock solution of CA was prepared in dimethyl sulfoxide (DMSO) at 10 mM and stored at -20 °C until use. Cell Counting Kit-8 (CCK-8) was from Dojindo Molecular Technologies (Kumamoto, Japan). CFDA-SE Cell Proliferation Assay and Tracking Kit was purchased from Beyotime Institute of Biotechnology (Jiangshu, China). VEGF, sodium heparin, Hochest 33258, and PTK787 were from Sigma (St. Louis, MO). Growth factor reduced Matrigel; Annexin V-FITC/PI Apoptosis Detection Kit was from BD Biosciences (San Jose, CA, USA). Specific rabbit polyclonal antibodies to endothelial nitric oxide synthase (eNOS), phospho-eNOS (Ser1177), AKT, phospho-AKT (Ser473), p38, phospho-p38 (Thr180/Tyr182), c-Raf, phospho-c-Raf (Ser338), Erk1/2, phospho-Erk1/2 (Thr202/Tyr204) and β-actin (as a loading control), U0126 (a MEK inhibitor), and LY294002 (a PI3K inhibitor) were from Cell Signaling Technology (Danvers, MA, USA). A hemoglobin human ELISA kit and monoclonal antibodies against VEGF and CD31 were from Abcam (Cambridge, UK). A human VEGF ELISA Kit was from eBioscience (San Diego, CA). All of the other chemicals were from Invitrogen (Carlsbad, CA) or otherwise indicated.
3
Matrigel Plug Assay for Angiogenesis
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Effect of CA on VEGF-stimulated angiogenesis in vivo was determined by Matrigel plug implantation assay which was based on the method of Wake et al. [28] . Briefly, the 5week-old male Kunming mice (25-28 g) were subcutaneously injected with 0.5 mL of liquid Matrigel (BD Bioscience) mixture containing 100 ng/mL VEGF and 100 units/mL heparin. Then the mice were treated daily for 2 weeks with intraperitoneal injections of either vehicle (4% tween-80 in saline) or CA (100 mg/kg in the vehicle). After the 15th day, the mice were euthanized, and the Matrigel plugs were excised and photographed. Then a portion of Matrigel plugs was homogenized in PBS (pH 7.4) and centrifuged at 10,000 rpm for 10 min. The concentration of Hb in the supernatant was measured at 450 nm directly using a hemoglobin human ELISA kit (Abcam).
4
Quantifying Free Hemoglobin in Plasma
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Plasma samples were thawed on ice and the Human Hemoglobin ELISA kit (Abcam, Cambridge UK) was applied to measure the free human haemoglobin concentration according to the instructions supplied by kit booklet.
5
Plasma Hemoglobin Quantification Protocol
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Plasma hemoglobin (Hgb) levels were determined using a Human Hemoglobin ELISA Kit (Abcam; cat# ab157707) as per manufacturer instructions using a 1:1000 dilution of plasma.
6
Red Blood Cell ATP Quantification
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The squeezed RBCs samples are centrifuged at 3,000 × g for 10 min, at 4°C. Supernatants are aspirated to measure the free hemoglobin concentration (CFHB) using Human Hemoglobin ELISA Kit (Abcam Co.). The CFHB total is 45%*CFHB /2% and 45% is normal hematocrit. To measure ATP in RBCs, the squeezed RBCs sample pellets are collected by centrifugation of 3,000 × g for 10 min 4°C and then assayed to determine the cellular ATP concentration (CATP total) using a Luminescent ATP detection assay kit (Abcam Co.). And the CATP Average is CATP total /N, and N is the number of RBCs counted by the cell count plate (Vic-Science Co). The ATP levels are calculated by total ATP concentration (CATP total) of RBCs samples and numbers of normal erythrocytes, echinocytes, spherocytes in N = 0, 5,000, 15,000 and 20,000. Ax + By + Cz = CATP total, where A, B, and C are the number of normal erythrocytes, echinocytes, and spherocytes, respectively, CATP total is the total ATP concentration of the RBCs, and x, y, and z are the average ATP levels of the normal erythrocyte, echinocyte, and spherocyte. In order to count number of RBCs samples, samples are applied to the edge of the chamber of cell count plate (Vic-Science Co.) between the coverslip and the V-shaped groove in the chamber. RBCs are allowed to be drawn into the chamber by capillary action. Stand for 2 min and then count the number of RBCs.
7
Quantifying Total Alpha-Synuclein in Plasma
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Total plasma alpha-synuclein levels were assessed using the U-PLEX Plus Human alpha-synuclein kit (Mesoscale Discovery, Cat. No. K151WKP-2) as previously described21 (link) using 1:8 dilution of the plasma samples. An 8-point calibration curve and 3 quality controls provided in the kit were included in each assay plate. The calibrator curve range was 0–10,500 pg/mL and was used to interpolate concentrations of total alpha-synuclein from plasma samples and quality controls. As plasma hemoglobin (Hgb) levels are important to include as a covariate in the analysis of total alpha synuclein in plasma samples21 (link), Hgb levels were also measured using a Human Hemoglobin ELISA Kit (Abcam; cat# ab157707) at a 1:1000 dilution of plasma, as per the manufacturer instructions. Hgb concentrations were interpolated from a 6-point calibration curve ranging from 0 to 200 ng/mL using a 4-parameter logistic curve fit. Inter-assay CV% for total alpha-synuclein and Hgb were < 15% and < 10% respectively.
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