VEGF protein levels produced by Cyp1b1+/+ and Cyp1b1-/- LSEC were determined using a mouse VEGF Immunoassay kit (R&D Systems). Cells were plated at 8×105 on 60 mm tissue culture dishes in EC growth medium. After 24 h, the cells were rinsed once with serum-free DMEM and incubated with 2 ml of serum-free DMEM for another 48 h. The conditioned medium was collected, centrifuged for 5 min at 400 xg to remove cell debris, and the secreted VEGF was analyzed per manufacturer’s instructions. The amount of VEGF was determined using a standard curve generated with known amounts of VEGF in the same experiment.
Mouse vegf immunoassay kit
Manufactured by R&D Systems
Sourced in Japan
The Mouse VEGF Immunoassay kit is a quantitative sandwich enzyme immunoassay designed for the measurement of mouse vascular endothelial growth factor (VEGF) in cell culture supernates, serum, and plasma.
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9 protocols using mouse vegf immunoassay kit
1
VEGF Secretion in Cyp1b1 Knockout Cells
2
VEGF Secretion Quantification in Astrocytes
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The level of VEGF secreted was assessed using a Mouse VEGF Immunoassay kit (R&D Systems) as we previously described [18 (link)]. Astroglial cells (6 x 105) were plated on a 60 mm tissue culture dish. Once the cells had reached ~90% confluence they were rinsed with serum-free DMEM and then grown in serum-free DMEM for 2 days. The conditioned medium was then clarified. The VEGF Immunoassay was performed in triplicate for the samples and normalized to the number of cells. A standard curve was generated to assess the sample VEGF levels. The assay was repeated twice with similar results.
3
Quantifying Secreted VEGF in PC Cells
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The effect of calcitriol and its solvent on secreted VEGF from PC was determined using a mouse VEGF Immunoassay kit (R&D Systems). Cells were plated at 2 × 105 on 60‐mm tissue culture dishes; the next day, medium was changed to medium containing appropriate treatment for 48 hours. Cells were then rinsed once with serum‐free DMEM and incubated in 2 mL serum‐free PC medium for 48 hours. CM was collected and centrifuged for 10 minutes at 400×g to remove the debris, and the secreted VEGF level was determined by an enzyme‐linked immunosorbent assay enzyme‐linked immunosorbent assay (ELISA). For the cell‐associated VEGF level, cell lysate was collected and used in the ELISA assay. Fifty microliters of samples was used in the assay. The assay performed in triplicate and was normalized to the number of cells. To determine the amount of VEGF, a standard VEGF curve was prepared and used to determine VEGF concentration.
4
Quantifying VEGF in Retinal Extracts
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The levels of VEGF were evaluated in retina extracts prepared from P15 Vdr +/+, Vdr +/-, and Vdr -/- mice using the Mouse VEGF Immunoassay Kit (R&D Systems, Minneapolis, MN). Briefly, retinas from each mouse were dissected and placed in 0.5 mL of PBS and stored at -80°C prior to analysis. VEGF levels were determined as recommended by the supplier.
5
VEGF Secretion by RPE Cells
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The level of VEGF produced by WT, TSP1−/−, and PEDF−/− RPE cells were determined using the Mouse VEGF Immunoassay Kit (R& D Systems). Cells (1 × 106) were plated in 60‐mm tissue culture dishes and allowed to reach 90% confluence. The cells were washed with serum‐free DMEM and incubated with 2 mL of growth medium without serum for 2 days. The CM were collected, centrifuged at 400 × g for 5 min to remove cell debris, and used for VEGF measurements as recommended by the supplier.
6
VEGF Secretion Regulation by Calcitriol
7
VEGF Quantification in Muscle Tissue
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VEGF concentrations were determined from gastrocnemius samples from Phd2f/f and Phd2 cKO mice using a mouse VEGF Immunoassay kit (R&D system, Japan). Briefly, muscle samples were prepared from gastrocnemius homogenates in 1.5 ml of ×1 PBS. The homogenates were subjected to two freeze-thaw cycles to break the cell membranes and centrifuged for 5 min at 5000×g. The VEGF immunoassay was carried out according to the manufacturer’s protocol.
8
VEGF Quantification in Mouse Retina
9
Quantifying Choroidal EC VEGF Secretion
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The amount of secreted VEGF produced by TSP1+/+ and TSP1−/− choroidal EC was determined using Mouse VEGF Immunoassay kit (R&D system). Cells (6×105) were plated on 60 mm tissue culture dishes and allowed to reach approximately 90% confluence. The cells were then rinsed once with serum free DMEM and incubated with 2 ml of EC growth medium without serum for 2 days. The CM was centrifuged to remove cell debris and the secreted VEGF in CM was analyzed according to manufacturer’s instruction. The amount of VEGF was determined using a standard curve generated with known amounts of VEGF in the same experiment.
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