After 13-day treatment of SMI, subcutaneous xenografts from Balb/c mice were removed, cut into small pieces and digested into single cell suspension by incubating in DMEM containing 2 mg/mL collagenase Type I (Sigma–Aldrich, Darmstadt, Germany) and 0.1 mg/mL DNase I (Roche, Basel, Switzerland) for 1 h. Then cells were strained with a 40 μm nylon mesh to remove cell clumps. Cells was incubated with phycoerythrin (PE) Rat Anti-Mouse CD31 (BD Biosciences) for 1 h. Anti-R-PE Magnetic Particles (BD Biosciences) was used as the secondary antibody. The endothelial cell population in the single cell suspension was enriched by magnetic isolation and washed with buffer (BD Biosciences) for 3 times. To identify the purity of endothelial cells, cells were incubated with APC Rat Anti-Mouse CD45 (BD Biosciences) and Fixable Viability Stain 440 V (BD Biosciences). Flow-cytometry was performed on a FACSAria II SORP (BD Biosciences).
Pe rat anti mouse cd31
Manufactured by BD
Sourced in United Kingdom, United States
The PE rat anti-mouse CD31 is a laboratory reagent used for the detection and analysis of the CD31 (Platelet Endothelial Cell Adhesion Molecule-1) protein in mouse samples. CD31 is a cell surface marker expressed on endothelial cells and is commonly used to identify and study vascular structures.
Automatically generated - may contain errors
Lab products found in correlation
Apc rat anti mouse cd45, by BD (2 mentions)
Dnase 1, by Roche (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Pe rat anti mouse cd45, by BD (2 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Facsaria 2 sorp, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Observer z1 microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by BD (1 mentions)
Pe rat anti mouse cd144, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Collagenase a, by Roche (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Apc mouse anti human cd90, by BD (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
9 protocols using pe rat anti mouse cd31
1
Isolation and Purification of Endothelial Cells from Xenograft Tumors
2
Flow Cytometric Analysis of Mouse Endothelial Cells
3
Endothelial Cells Phenotyping and Imaging
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5 × 104 ECs were cultured in 24-well plates for 24 h and fixed with 10% formalin for 10 min and blocked with blocking buffer containing 5% bovine serum albumin (BSA, bioWORLD) in 1X DPBS to block non-specific binding sites for 1 h at RT. The cells were then stained with either 0.01 mg/ml PE Rat Anti-Mouse CD31 (553373, BD Biosciences) or 0.01 mg/ml PE Rat Anti-Mouse CD144 (562243, BD Biosciences) antibodies in a staining buffer containing 1% BSA in 1X DPBS and incubated at 4°C overnight. The cell nuclei were stained with 1:5000 DAPI for 5 min and washed 3 times. Then images were taken using a Zeiss Observer Z1 microscope.
4
Isolation and Analysis of Retinal Endothelial Cells
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Retinas from neonatal P7 mice were dissected in cold PBS and rinsed in PBS. Retinal cells were dissociated with 1 mg/ml Collagenase A (Roche, Germany, 10103578001), 3 U/ml DnaseI (Roche) in DMEM (Thermo Fisher Scientific) at 37°C for 30 min and cell suspension was passed through cell strainer. After several washes in PBS, cell suspension was incubated with PE rat anti-mouse CD31 (BD Biosciences, 553373) and APC rat Anti-Mouse CD45 (BD Biosciences,561018) antibodies on ice for 15 min. Cells were then washed and applied to FACS. RNA from CD31+CD45- cells was extracted using QIAGEN microRNA kit (Netherlands). PCR for the different Wnt ligands were performed with standard PCR protocols using primers listed in Supplementary file 2 .
5
Xenograft Tumor Angiogenesis Inhibition
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Example 4
Xenogaft tumors from the MCF-7 and MDA-MB-231 models were tested for inhibition of angiogenesis by HBB using CD31 staining.
Staining Protocol
The samples were cryosectioned at 6 um and fixed with chilled 100% acetone for 10 minutes. Samples were blocked with 3% donkey serum in PBS for 30 minutes at 37 C.
The antibody used to stain CD-31 was from BD Pharmingen PE Rat anti Mouse CD31, catalog number 553373. It was used at a dilution of 1:50 in blocking buffer for 1 hour at 37 C.
The samples were then stained for 10 minutes with DAPI at a concentration of 1:2000, from Life Technologies, catalog number D1306.
Imaging Samples
Each sample was imaged in 4-5 areas. There were 3 samples per condition, PBS vs HBB. The data analyzed were found to be statistically significant with a p-value of less than 0.01.
HBB inhibited angiogenesis in both models but tumor growth wasn't inhibited in the MDA-MB-231 model, only in the MCF-7 model (
6
Flow Cytometric Analysis of Porcine ASCs
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The isolated ASCs' surface antigen expression was analyzed by flow cytometry according to a previously reported method [23] (link). One million passage 3 ASCs were suspended in 100 μL PBS containing 10 μg/ml fluorescein isothiocyanate-conjugated primary antibodies specific to mesenchymal stromal cells (MSCs) (CD29, CD44, CD90, and CD105) and hematopoietic cells (CD31 and CD45) (n = 3). The following expression markers reactive with the porcine antigen isoforms were used: Alexa Fluor 647 Mouse Anti-Pig CD29 (BD Bioscience, New Jersey, USA), Anti-CD44 antibody [IM7] (Abcam plc, Cambridge, UK), APC Mouse Anti-Human CD90 (BD-Biosciences), Anti-CD105 antibody [MEM-229] (Abcam plc), PE Mouse Anti-Rat CD31 (BD Biosciences), and Monoclonal Antibody to CD45/LCA (CD45R)-PE (Acris Antibodies, Inc. CA, USA) [24] (link), [25] (link), [26] (link), [27] (link), [28] (link). Cell fluorescence was evaluated with a Gallios flow cytometer (Beckman Coulter, Tokyo, Japan) and the data were analyzed using Karuza for Gallios software (Beckman Coulter).
7
Endothelial Cell Differentiation and Angiogenesis
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Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (D-MEM/F-12, Gibco, USA), bovine serum albumin (BSA), and trypsin/EDTA (Gibco-BRL); EGM-2 Bullet Kit (mixture of ascorbic acid, hydrocortisone, EGF, IGF-I, heparin, VEGF, and FGF2,; Lonza); insulin-transferrin-selenium sodium pyruvate (ITS; Invitrogen); Jagged1 Fc Chimera (599-JG; R&D Systems); rat immunoglobulin G (IgG) (I4131; Sigma Aldrich); PE-mouse anti-Rat CD31 (BD Pharmingen™); anti-Hif-1α antibody (Proteintech); R-PE-conjugated Donkey Anti-Goat IgG(H + L) (Proteintech); anti-Jagged-1 antibody (Affinity); anti-N1ICD antibody (CST); anti-Hey1 antibody (Abcam); anti-VEGF receptor2 antibody (Abcam); anti-Von Willebrand factor (vWF) antibody (Abcam); goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen); TRIzol (Takara, Biotechnology, Dalian, China); 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes); reverse Q-PCR kit, and RT-PCR kit, protein lysis buffer kit (Beyotime Biotechnology); anti-actin antibody (Sigma); horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG (Beyotime Biotechnology); Matrigel (Corning); Dual-GLO Luciferase Assay System (Promega); Fugen6 (Roche).
8
Single-cell RNA-seq of Immune Cells
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The cells were subjected to staining using the 7-AAD Viability Staining Solution (Invitrogen, 00-6993-50) and lineage markers, rat anti-mouse CD11b-PE (BD Pharmingen, 557397), rat anti-mouse CD31-PE (BD Pharmingen, 553373), rat anti-mouse CD45-PE (BD Pharmingen, 553081), biotin rat anti-mouse TER-119 (BD Pharmingen, 553672), and streptavidin-PE (BD Pharmingen, 554061). The cells were then allowed to pass through a 35-μm cell strainer (Corning, 352235). Single cells were sorted using SH800 (SONY, Tokyo, Japan) in 2% bovine serum albumin/PBS. Sorted cells were subjected to processing using the Chromium Controller (10x Genomics, Pleasanton, CA, USA), Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000128), and MGIEasy Universal Library Conversion Kit (App-A) (MGI, Shenzhen, China) following the manufacturer’s instructions. The library was sequenced using DNBSEQ-G400 (MGI).
9
Skeletal Muscle Stem Cell Isolation
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For isolation of SCs by FACS sorting, skeletal muscles from hind limbs were prepared similarly as for flow cytometry analysis, resuspended in PBS + 2% FBS, and then incubated with the following antibodies for 30 min on ice: rat anti-mouse α7integrin-APC (1:15, 334,908, R&D Systems), rat anti-mouse CD34-FITC (1:30, RAM34, eBioscience), rat anti-mouse CD45-PE (1:30, 30-F11, BD Biosciences), rat anti-mouse CD31-PE (1:30, MEC13.3, BD Biosciences), and rat anti-mouse Sca-1-PE-Cy7 (1:30, D7, eBioscience). After incubation, cells were washed, filtered through a 40-μm cell strainer, and resuspended in PBS + 2% FBS with Hoechst 33342 (10 μg/ml) and 7-AAD (1:40, BD Biosciences). Cells were sorted with MoFlo XDP (Beckman Coulter) cell sorter.
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