CD8+ T lymphocytes were purified from splenocytes using a CD8+ isolation kit (Miltenyi Biotec). Purified CD8+ T lymphocytes were cultured in the presence of plate-bound anti-CD3 (10 µg/ml) with or without recombinant murine VEGF-A (50 ng/ml; Miltenyi Biotec). After 48 h of culture, cells were harvested and analyzed by cytometry or used to extract mRNA. In some experiments, anti–VEGF-R1 (20 µg/ml; R&D Systems) or anti–VEGF-R2 (10 µg/ml; clone 91202; R&D Systems) antibodies or isotype control were added to the culture medium. In some experiments, 11R-VIVIT (Merck Millipore) was added 1 h at 5 µM before the addition of VEGF-A and during the stimulation with VEGF-A.
Anti vegfr1
Manufactured by R&D Systems
Sourced in Japan
Anti-VEGFR1 is a monoclonal antibody that binds to and inhibits the activity of the vascular endothelial growth factor receptor 1 (VEGFR1).
Automatically generated - may contain errors
Lab products found in correlation
Cd8 isolation kit, by Miltenyi Biotec (1 mentions)
Anti vegfr2, by R&D Systems (1 mentions)
11r vivit, by Merck Group (1 mentions)
Vegf a, by Merck Group (1 mentions)
Anti cd3, by Miltenyi Biotec (1 mentions)
Anti fcr monoclonal antibody 2.4g2, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Dako envision dual link system hrp, by Agilent Technologies (1 mentions)
Anti vegfr 2 antibody, by R&D Systems (1 mentions)
Anti ng2, by Merck Group (1 mentions)
Fitc conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Facscan caliber flow cytometer, by BD (1 mentions)
Anti pdgfrα, by Thermo Fisher Scientific (1 mentions)
Vegfr2, by R&D Systems (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
4 protocols using anti vegfr1
1
VEGF-A Modulation of CD8+ T Cell Activity
2
Quantification of VEGFR1-Positive Cells
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Blood was drawn from the tail vein 48 h after reperfusion. The white blood cell fraction, including platelets, was obtained by separation on Ficoll and analyzed by flow cytometry, as previously described [25] (link). Briefly, cells were labeled with phycoerythrin-labeled anti-VEGFR1 (R&D Systems, MN) and PerCP-Cy5.5-labeled anti-CD11b (LifeSpan Biosciences Inc., WA) antibodies in the presence of an anti-FcR monoclonal antibody (2.4G2; BD Biosciences). After washing, the cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences) and small cells (with low forward scatter [FSC]) were gated for peripheral blood analysis. The percentage of VEGFR1-positive cells was calculated from the flow cytometry results.
3
Immunohistochemical Analysis of VEGF Signaling
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Formalin fixed tissue was stained by routine H-E staining or immunohistochemistry (IHC). The following primary antibodies were used for IHC: anti-VEGF-A antibody (Merck Millipore Japan, Tokyo, Japan), anti-VEGFR-1 (R&D Biosystems, Minneapolis, MN, USA), and anti-VEGFR-2 antibody (R&D Biosystems). All primary antibodies were incubated with tissues at a 1:100 dilution for 60 min. The secondary antibody Dako EnVision™+ Dual Link System-HRP (Dako, Tokyo, Japan) was then incubated with the tissues for 15 min at room temperature. Protein expression was visualized using a 3,3′-diaminobenzidine tablet (Dako).
4
Flow Cytometric Profiling of Astrocytes
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Flow cytometry was performed as we previously described [18 (link), 23 (link)] by rinsing dishes with phosphate buffered saline (PBS) containing 0.04% EDTA followed by an incubation with 1.5 ml of cell dissociation solution (Tris-buffered saline (TBS) (20 mM Tris-HCl and 150mM NaCl; pH 7.6) TBS containing 2 mM EDTA and 0.05% BSA) to remove the cells from the plate. Next, astrocytes were rinsed with TBS then blocked in TBS with 1% goat serum on ice for 20 min. This was followed by incubation with the appropriate primary antibody including anti-GFAP (Dako #Z0334), anti-PDGF-Rα (eBiosciences #14140182), anti-NG2 (Millipore, Temecula, CA #AB5320), anti-VEGFR1 (R&D Systems #MAB471) and VEGFR-2 (R&D Systems #MAB443). The astrocytes were washed twice and then incubated with the appropriate FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) diluted in TBS with 1% BSA and incubated on ice for 30 min. The resulting stained astrocytes were washed twice and resuspended in 0.5 ml of TBS with 1% BSA. The samples were analyzed with a FACScan caliber flow cytometer (Becton-Dickinson) and the resulting representative histograms are shown.
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