Total proteins were extracted from CZ by using the Minute™ total protein extraction kit (Invent Biotechnologies). Quantification of total proteins was achieved by using the Pierce™ BCA™ Protein-Assay (Thermo Fisher Scientific). Measurements of protein concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were performed by using the Rat GM-CSF ELISA Kit (Assay Genie, RTFI00020), MCP1 (CCL2) Rat ELISA Kit (Abcam, ab100778) and VEGF Rat ELISA Kit (Abcam, ab100787), respectively. Concentrations were measured spectrophotometrically by light absorbance using the Spark multimode microplate reader (Tecan Group AG). All samples were run in triplicate. The final concentration was expressed as pg/μg total protein.
Rat vegf elisa kit
Manufactured by Abcam
Sourced in United Kingdom, Japan
The Rat VEGF ELISA kit is a quantitative sandwich enzyme immunoassay designed for the measurement of rat vascular endothelial growth factor (VEGF) levels in cell culture supernatants, serum, and plasma samples.
Automatically generated - may contain errors
Lab products found in correlation
Minute total protein extraction kit, by Invent Biotechnologies (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Elx800 absorbance microplate reader, by Agilent Technologies (1 mentions)
Apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Sirt1, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Notch1, by Abcam (1 mentions)
Rat il 6 elisa kit, by Abcam (1 mentions)
Rat tnf alpha elisa kit, by Abcam (1 mentions)
7 protocols using rat vegf elisa kit
1
Quantifying Inflammatory Cytokines in Rat Tissue
2
Quantifying Ocular Angiogenic Factors
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From the extracted protein of the eyes, we measured the protein expression of vascular endothelial growth factor (VEGF) using a VEGF rat ELISA kit (abcam).
For the protein expressions of CD31 and endothelial nitric oxide synthase (eNOS), we performed Western blot analysis. The extracts were separated by NuPAGE 4% to 12% Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene fluoride membrane. Before the primary antibody reaction, the membranes were cut around molecular weight of each targeted molecule. Primary antibodies against CD31 (1:2000; abcam), eNOS (D9A5L; rabbit monoclonal; 1:1000; Cell Signaling Technology, Danvers, MA, USA), and α-tubulin (rabbit monoclonal; 1:8000; abcam) were used. Immunoreactive bands were detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). The expression levels of all proteins were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to that of α tubulin.
For the protein expressions of CD31 and endothelial nitric oxide synthase (eNOS), we performed Western blot analysis. The extracts were separated by NuPAGE 4% to 12% Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene fluoride membrane. Before the primary antibody reaction, the membranes were cut around molecular weight of each targeted molecule. Primary antibodies against CD31 (1:2000; abcam), eNOS (D9A5L; rabbit monoclonal; 1:1000; Cell Signaling Technology, Danvers, MA, USA), and α-tubulin (rabbit monoclonal; 1:8000; abcam) were used. Immunoreactive bands were detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). The expression levels of all proteins were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to that of α tubulin.
3
Secretion Analysis of Growth Factors
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MSCs were cultured in a normoxic condition of 95% air and 5% carbon dioxide or a hypoxic condition of 95% nitrogen and 5% oxygen at 37°C for 24 hours. To assess the secretion of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and plate-derived growth factor (PDGF), the conditioned medium was collected from wild type MSCs, vector-MSCs, and hBcl-xL-MSCs, respectively, and ELISA was performed using the VEGF Rat ELISA Kit (Abcam, Cambridge, Cambs, UK), PDGF-AA Rat ELISA Kit (Abcam, Cambridge, Cambs, UK), and IGF-1 Rat ELISA Kit (Abnova, Taibei, Taiwan) according to the manufacturers' protocol. The optic density (OD) value of each sample was read on an ELX-800 absorbance microplate reader (Biotek, Winooski, VT, USA) and adjusted with the TMB empty control. The concentration of each sample was calculated according to the standard curve.
4
Sirtuin-1 Regulation of Diabetic Retinopathy
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Angio-proteomie (Boston, MA, USA) provided human retinal endothelial cells (HRECs). Cell culture and transfection-related reagents were from Invitrogen (Carlsbad, CA, USA). SIRT1-siRNA (si-SIRT1-1/-2) and the negative control (si-NC) were from Sangon Biotech (Shanghai, China). The commercial kits for the measurement of glucose and triglyceride contents were from Boxbio (Beijing, China). The caspase-3 assay kit was from QCbio Science & Technologies (Shanghai, China). Sigma Aldrich (San Luis, MO, USA) provided streptozotocin (STZ) and the commercial kits for the measurement of reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), and inflammatory cytokines. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide (MTT) was from Aladdin (Shanghai, China). Apoptosis detection kit and western blotting analysis-related reagents were from Thermo Fisher Scientific (Waltham, MA, USA). Rat VEGF ELISA kit, the primary antibodies (Notch1, Hes1, Hey2, SIRT1, and GAPDH), and the HRP-conjugated secondary antibody were procured from Abcam (Cambridge, UK).
5
Prolactin and VEGF Measurement in Cell Cultures
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Amount of PRL in serum and CSF were measured by chemiluminescent enzyme immunoassay using PATHFAST® (LSI Medience Corporation, Tokyo, Japan) according to the manufacturer’s protocol. For these measurements, the clinical serum reference range was 4.98−26.4 ng/mL for males and 1.74−26.8 ng/mL for females. Amount of PRL in the conditioned medium of SDR-P-1D5 cells was measured by prolactin EIA Kit (A05101; Bertin Bioreagent, Montigny le Bretonneux, France), and in the conditioned medium of MSH-P3 cells was measured by pig prolactin ELISA KIT (MBS705496, MyBioSource, CA, USA). Amounts of vascular endothelial growth factor (VEGF) in the conditioned medium of SDR-P-1D5 cells was measured by rat VEGF ELISA KIT (ab100786; Abcam, Tokyo, Japan), and in the conditioned medium of MSH-P3 cells was measured by pig VEGF 164 ELISA KIT (ab218298; Abcam, Tokyo, Japan).
6
Co-culture of BMSCs and EPCs
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BMSCs and EPCs were co-cultured in direct contact at a 1:1 ratio or were cultured alone; for this, were seeded onto 24-well plates in conditioned medium. The cell culture supernatants were assessed by a rat VEGF ELISA kit (ab100786; Abcam, Japan) according to the instructions.
7
Assessing Inflammatory and Oxidative Markers
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Factors in the tissue supernatants or cell lysates were assayed using the rat IL-6 ELISA kit (ab234570, Abcam), rat TNF alpha ELISA kit (ab100785, Abcam), rat Superoxide Dismutase 1 (SOD1) ELISA kit (E4584, BioVision, Inc., Exton, PA, USA), and rat VEGF ELISA kit (ab100787, Abcam) according to the manufacturer's protocol. Malondialdehyde (MDA) levels in tissue supernatants or cell lysates were measured using the Lipid Peroxidation (MDA) Fluorescence Assay Kit (ab118970, Abcam, Ex/Em 532/553 nm).
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