Tumors were harvested 6 or 24 hours following FUS+MBs BTB/BBB opening. The tumors were minced and placed in digestion media containing 0.42U/ml Liberase TM (Roche). Samples were digested at 37 °C for 15 minutes and triturated every 5 minutes. Samples were homogenized (using glass homogenizers) and filtered through 70 µm filters. Subsequently, all samples were centrifuged at 1500 rpm for 15 minutes. The pellets were resuspended in CD45+ magnetic beads (Miltenyi Biotech) with Fc Block (1:1000) and incubated for 15 minutes at 4 °C. Samples were washed with AwesomeMacs Buffer and centrifuged at 1500 rpm for 5 minutes. Samples were separated with autoMACS Pro Separator with POSSEL AutoMACS protocol. The CD45 negative fraction was pelleted and stained with CD31 endothelial cell panel. Cells were Fc blocked and stained with fluorescent antibodies for CD31 (clone 390, eBioscience), CD45 (clone 30-F11, Biolegend), E-sel (clone 10E9.6, DB), P-selectin (clone RB40.34, DB), ICAM-1 (clone YNI/1.7.4, Biolegend), VCAM-1 (clone 429, BD) and Live/dead Aqua stain (eBioscience). Cells were fixed in 2% PFA.
Cd31 clone 390
Manufactured by Thermo Fisher Scientific
CD31 (clone 390) is a laboratory reagent used in flow cytometry and immunohistochemistry applications. It is an antibody that specifically binds to the CD31 antigen, which is expressed on the surface of endothelial cells and certain types of leukocytes. The primary function of this reagent is to facilitate the identification and enumeration of cells expressing the CD31 antigen.
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Lab products found in correlation
Cd45 magnetic beads, by Miltenyi Biotec (1 mentions)
Vcam 1, by BD (1 mentions)
Cd45 clone 30 f11, by BioLegend (1 mentions)
Liberase, by Roche (1 mentions)
Cd3 clone 145 2c11, by Thermo Fisher Scientific (1 mentions)
Ifnγ clone xmg1, by BD (1 mentions)
Cd19 clone 1d3, by BD (1 mentions)
Nk1.1 clone pk136, by BioLegend (1 mentions)
Facs aria machine, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cd45 clone 30 f11, by Thermo Fisher Scientific (1 mentions)
Zombie uv, by BioLegend (1 mentions)
Dmrxa fluorescent microscope, by Leica (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
4 protocols using cd31 clone 390
1
Isolation and Characterization of Tumor Endothelial Cells
2
Isolation and Culture of Intestinal Myeloid and Stromal Cells
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Isolation of intestinal myeloid and stromal cells were performed as previously described (Satoh-Takayama et al., 2008 (link); Stzepourginski et al., 2015 (link)). Cells were blocked and stained with indicated antibodies: CD3 (clone 145-2C11; eBioscience); CD19 (clone 1D3; BD); NKp46 (clone 29A1.4; eBioscience); IFN-γ (clone XMG1.2; BD); NK1.1 (clone PK136; Biolegend); and CD31 (clone 390; eBioscience). Syrian hamster antibody to gp38 was a gift from A. Farr (University of Washington, Seattle, WA).
Cells were analyzed with an LSRFortessa flow cytometer (BD) or sorted with a FACSAria machine (BD). Flow cytometry analysis was done with the FlowJo software (Tree Star). Sorted stromal CD45−gp38+ cells were grown in DMEM containing 10% fetal calf serum at 150,000 cells/well in 24-well plates coated with rat tail collagen (10 µg/cm2). Cells were treated 4 d after sorting with the mouse recombinant IL-17 (R&D; 421-ML) or mouse recombinant IL-23 (R&D; 1887-ML) for 24 h.
Cells were analyzed with an LSRFortessa flow cytometer (BD) or sorted with a FACSAria machine (BD). Flow cytometry analysis was done with the FlowJo software (Tree Star). Sorted stromal CD45−gp38+ cells were grown in DMEM containing 10% fetal calf serum at 150,000 cells/well in 24-well plates coated with rat tail collagen (10 µg/cm2). Cells were treated 4 d after sorting with the mouse recombinant IL-17 (R&D; 421-ML) or mouse recombinant IL-23 (R&D; 1887-ML) for 24 h.
3
Flow Cytometry Analysis of Lung Cell Populations
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Single suspensions of enzyme-digested lung tissue were used to perform flow cytometry as previously described 24 hours after retro-orbital injection of adult mice with np-mCherry-FOXF1 (labeled with DyLight) or npEmpty polyplexes (control mice).23 (link),24 (link) Live cells were identified with Zombie UV™ (BioLegend). Cells were evaluated for presence of CD45 (clone 30-F11; eBioscience) and CD31 (clone 390; eBioscience) for identification of immune cells (CD45+CD31–) and endothelial cells (CD31+CD45–). Cells not expressing any of these markers (CD45–CD31–) were then evaluated for epithelial cells using CD326 (clone G8.8; eBioscience). Cells not expressing any of these markers (CD45–CD31–CD326–) were then evaluated for pericytes (NG2+CD45–CD31–CD326–) using NG2 antibody (clone132.39; Millipore). Fibroblasts (CD140a+CD45–CD31–CD326 NG2–) were identified using CD140a (clone APA5; BD Biosciences). Stained cells were analyzed using 5 laser Cytek® Aurora (spectral system).
4
Immunofluorescent Staining of Mouse Tissue
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6-μm cryosections were fixed for 5 min in ice-cold acetone and air-dried for an additional 10 min. After blocking of Fc receptors with serum, sections were incubated with primary antibodies for 1 h at room temperature, followed by a 30-min incubation with a Fluor-Alexa-labeled conjugate (Molecular Probes). Sections were embedded in ProLong Gold (Invitrogen) and analyzed on a Leica DMRXA fluorescent microscope. The antibodies used were CD31 (clone 390; eBioscience), PNAd (Meca79; supernatant was provided by R. Mebius, VU University Medical Center Amsterdam, Amsterdam, Netherlands).
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