Flow cytometry was performed as described elsewhere (Notta et al., 2016 (link)). Briefly, fresh frozen tumor samples were mechanical dissociated sharply then suspended in 9 ml of RPMI supplemented with 1% FBS and 1ml of 10× collagenase/hylauronidase mix (Stemcell technologies), filtered through a 70-150μm nylon mesh, centrifuged, re-suspended in cryopreservation media (20% FBS/10% DMSO final) and stored at −150 °C. Viable cells were then thawed, spun at ~ 1,000 r.p.m. for 20 min at 4 °C, and re-suspended in 100 μl of PBS + 5% FBS for antibody staining and cell sorting on the BD FACSAria III using 4-laser configuration. The following antibodies were used for cell sorting: GlyA FITC (BD bioscience, clone HIR2), CD140b PE (BD bioscience, clone 28D4), CD45 PC5 (Beckman Coulter, clone IM1833), EpCAM PerCP-eFluor710 (eBioscience, clone 1B7), CD31 PC7 (eBioscience, clone WM-59), CD90 (BD Biosciences, clone 5E10), CD34 APC7 (BD bioscience, clone 581, custom conjugation).
Cd140b pe
Manufactured by BD
Sourced in United States, United Kingdom
CD140b-PE is a fluorescently-labeled monoclonal antibody that binds to the CD140b cell surface protein. It is commonly used in flow cytometry applications to identify and analyze cells expressing CD140b.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 pc5, by Beckman Coulter (1 mentions)
Facsaria 3, by BD (1 mentions)
Cd31 fitc, by BD (1 mentions)
Cd146 pe, by BD (1 mentions)
Cd105 percp cy5, by BD (1 mentions)
Cd117 pe, by BD (1 mentions)
Cd34 fitc, by BD (1 mentions)
Cd25 pe, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Cd45 fitc, by BD (1 mentions)
Cd90 fitc, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Calibur flow cytometer, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Tritc labeled goat polyclonal anti rabbit igg, by Merck Group (1 mentions)
Fluoroshield mounting medium with dapi, by Merck Group (1 mentions)
Cd144 fitc, by BD (1 mentions)
4 protocols using cd140b pe
1
Multicolor Flow Cytometry for Tumor Cell Isolation
2
Comprehensive Flow Cytometry Analysis of USCs
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Flow cytometry analysis for the USCs involved staining the USCs with specific labeled anti-human antibodies: CD25-PE, CD31-FITC, CD34-FITC, CD44-FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PerCP-CY5.5, CD117-PE, CD140b-PE, and CD146-PE (BD Pharmingen™). Briefly, USCs (p4) were trypsinized, and 5.0 × 105 cells were washed with pre-chilled PBS containing 1% bovine serum albumin (BSA). The fluorescence conjugated antibodies listed above were incubated with USCs on ice for 30 min in the dark. IgG1-PE, IgG1-FITC, IgG2b-FITC, and IgG1-PerCP-CyTM5.5-conjugated isotype control antibodies were used to determine background fluorescence. Cells were washed twice with wash buffer, passed through a 70 µm filter, and analyzed using FACSCalibur™ flow cytometry (BD Biosciences, Franklin Lakes, NJ, United States).
3
Quantifying ASC-EC Coculture Ratios
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EC‐ASC cocultures were harvested, cell numbers were determined using hemocytometer and cell suspensions were incubated with CD140b‐PE (ASC marker) and CD31‐APC (EC markers) IgG (BD, San Diego, CA, USA) on ice for 20 min. ASC:EC ratios were determined using Calibur flow cytometer and Cell QuestPro software (BD).
4
Immunocytochemical Staining of Vascular Cells
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The immunocytochemical staining was performed as described earlier (Huttala et al., 2015) (link) except that the fixative used here was 4% formaldehyde at room temperature for 20 min. Antibodies used were anti-von Willebrand factor IgG (produced in rabbit, Sigma), anti-collagen IV IgG (produced in mouse, Sigma), FITC-labeled goat polyclonal anti-mouse IgG (Sigma), and TRITC-labeled goat polyclonal anti-rabbit IgG (Sigma). In addition, for confocal imaging, anti-collagen IV (produced in rabbit, ab6586, Abcam, Cambridge, UK) used with secondary antibody TRITC-labeled goat polyclonal anti-rabbit IgG (Sigma), CD140b-PE (BD Biosciences, 558821), and CD144-FITC (BD Biosciences, 560411) were used. Nuclei were stained using Flu-oroshield™ with DAPI mounting medium (Sigma).
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