Plasma samples were obtained from the blood based perfusate by centrifugation using standard protocol and snap frozen in liquid nitrogen for further analysis (long-term preservation at -80 C°). Free haemoglobin was measured spectrometrically assay using Drabkin solution (D5941, Sigma). The cytokines level in the plasma were analysed using LEGENDplex™ Human Inflammation Panel (740118, Biolegend). 50 μL of plasma were assayed following the manufacture’s indications. In human livers 7-10, the upper measurable limit of the analytical method was taken for statistical analysis, because this value was exceeded. 8-OHDG (KA0444, Abnova) for DNA damage and Cytochrome C (CSB-EL006328PI, Cusabio) for mitochondrial injury were measured with ELISA according to manufacturer recommendation.
Drabkin solution
Manufactured by Merck Group
Drabkin solution is a reagent used for the colorimetric determination of hemoglobin concentration in blood samples. It is a standardized solution that contains potassium ferricyanide, potassium cyanide, and a buffer. When mixed with a blood sample, the solution converts hemoglobin into cyanmethemoglobin, which can be measured spectrophotometrically.
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Lab products found in correlation
Csb el006328pi, by Cusabio (2 mentions)
Legendplex human inflammation panel, by BioLegend (2 mentions)
Multiskan go, by Thermo Fisher Scientific (1 mentions)
Dispase, by BD (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase a, by Roche (1 mentions)
Matrigel, by BD (1 mentions)
Matrigel, by Merck Group (1 mentions)
Vegf a, by R&D Systems (1 mentions)
Tumor necrosis factor α tnf α, by R&D Systems (1 mentions)
Matrigel, by R&D Systems (1 mentions)
Matrigel, by Roche (1 mentions)
4 protocols using drabkin solution
1
Plasma Biomarkers of Liver Injury
2
Plasma Biomarkers for Liver Assessment
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Plasma samples were obtained from the blood-based perfusate by centrifugation using standard protocol and snap frozen in liquid nitrogen for further analysis (long-term preservation at −80 °C). Free hemoglobin was measured spectrometrically assay using Drabkin solution (D5941, Sigma). The cytokine levels in the plasma were analyzed using LEGENDplex Human Inflammation Panel (740118, Biolegend). Fifty microliters of plasma were assayed following the manufacturer’s instruction. In human livers 7 to 10, the upper measurable limit of the analytical method was taken for statistical analysis, because this value was exceeded. 8-Oxo-2′-deoxyguanosine (KA0444, Abnova) for DNA damage and cytochrome C (CSB-EL006328PI, Cusabio) for mitochondrial injury were measured with enzyme-linked immunosorbent assay according to the manufacturer’s recommendation.
3
Hemoglobin Quantification in Mouse Blood
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Two microliters of mouse tail blood were collected daily and mixed with 498 µL of Drabkin solution (1 mM potassium ferrocyanide, 7.6 mM potassium cyanide and 11.9 mM sodium bicarbonate (Sigma-Aldrich)). The mixture was incubated at room temperature for 5 min. The absorbance was measured at 540 nm on a plate reader (Multiskan GO, Thermo Fisher Scientific, Inc., Waltham, MA, USA). To calculate the hemoglobin concentration, a curve was prepared with a rat hemoglobin standard (Sigma-Aldrich).
4
Angiogenic Potential of Conditioned Media
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The indicated CMs, mixed with heparin (PharmaTex, Milan, Italy) and 500 μl Matrigel® (BD Biosciences) were injected subcutaneously in C57/BL6 mice or NSG mice, when directly specified. Groups of four mice (two plugs per mouse) were used for each experiment. The CMs were replaced by medium supplemented or not with VEGF-A (100 ng/ml) and Tumor Necrosis Factor α (TNFα; 2 ng/ml) (R&D Systems, Inc. MN, USA) in the positive and negative controls, respectively. Four days after injection, the Matrigel plugs were recovered and processed for hemoglobin quantization using Drabkin solution (Sigma-Aldrich). The optical density of each sample was determined at 540 nm. Results were normalized for 100 mg of Matrigel®.
When specified, CMs were pre-incubated for 30 minutes in presence of 1 μg/ml of the blocking antibody against VEGF-A (clone MAB293; R&D systems) before injection.
For the analysis of cell infiltrate, the recovered Matrigel® plugs were dissociated mechanically in serum-free medium containing DNase I (Roche), collagenase A (Roche) and dispase (BD Biosciences) and then incubated for 30 min at 37 °C under gentle agitation. The obtained cells were analysed by flow cytometry (see ‘Immunofluorescence' section).
When specified, CMs were pre-incubated for 30 minutes in presence of 1 μg/ml of the blocking antibody against VEGF-A (clone MAB293; R&D systems) before injection.
For the analysis of cell infiltrate, the recovered Matrigel® plugs were dissociated mechanically in serum-free medium containing DNase I (Roche), collagenase A (Roche) and dispase (BD Biosciences) and then incubated for 30 min at 37 °C under gentle agitation. The obtained cells were analysed by flow cytometry (see ‘Immunofluorescence' section).
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