To obtain lung endothelial cells, the lung tissues were digested with Collagenase I (Roche, 11088793001) plus Dispase II (Roche, 04942078001) at a working concentration of 2 mg/mL for 30 min at 37 °C. Red blood cells were then disintegrated using red blood lysis buffer (155 mM NH4Cl,12 mM NaHCO3, 0.1 mM EDTA). The remaining viable cells were incubated with PerCP-Cy™5.5 Anti-Mouse CD45 antibody (BD, 550994) and PE Rat Anti-Mouse CD31 antibody (BD, 553373) at 4 °C under gentle rotation for 30 min. After centrifugation, the cells were washed once with PBS containing 1% donkey serum and 2 mM EDTA. Finally, flow cytometry sorting was performed to obtain CD45−CD31+ lung endothelial cells with FACS Aria Ⅲ (BD Biosciences).
Percp cy5.5 anti mouse cd45 antibody
Manufactured by BD
Sourced in United States
The Percp-Cy5.5 anti-mouse CD45 antibody is a fluorescent-conjugated monoclonal antibody that binds to the CD45 surface marker on mouse cells. It can be used for the identification and analysis of mouse leukocytes in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2 cell sorter, by BD (2 mentions)
Facsaria 3, by BD (1 mentions)
Pe rat anti mouse cd31 antibody, by BD (1 mentions)
Dispase 2, by Roche (1 mentions)
Collagenase 1, by Roche (1 mentions)
Complete 1 640 medium, by Solarbio (1 mentions)
Ghost dyetm red 780, by Cytek Biosciences (1 mentions)
Ack buffer, by BD (1 mentions)
Collagenase 4, by Solarbio (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Chromium controller, by 10x Genomics (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Agilent 4200, by Agilent Technologies (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Facsaria 2 sorp, by BD (1 mentions)
Ls003119, by Worthington (1 mentions)
Pe anti mouse acsa 2, by Miltenyi Biotec (1 mentions)
Brilliant violet 605 anti mouse cd31, by BioLegend (1 mentions)
Fitc anti mouse human cd11b antibody, by BioLegend (1 mentions)
Percoll density gradient, by GE Healthcare (1 mentions)
4 protocols using percp cy5.5 anti mouse cd45 antibody
1
Isolation of Lung Endothelial Cells
2
Isolation of Lung CD45+ Cells
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Single cell suspensions were isolated from mouse lungs by collagenase digestion following recently published protocols (Reyfman et al., 2019 (link)). Briefly, mice were anesthetized with an overdose of 0.5% pentobarbital, then the lungs were perfused with 1 mL complete 1,640 medium (Solarbio) with 10% FCS (Hyclone) containing collagenase IV (Solarbio) and Dnase I (Sigma) through the trachea, chopped with scissors, and subsequently incubated for 20 min at 37°C with mild agitation. The resulting lung homogenate was passed through a 40-μm filter and resuspended in ACK buffer (BD) for 15 min on ice. Then, the cells were centrifuged at 400 g for 6 min and incubated with Percp-Cy5.5 anti-mouse CD45 antibody (BD) and Ghost DyeTM Red 780 (Tonbo) for 15 min on ice. BD FACS ARIA II cell sorter was applied to sort CD45+ cells from lung cell suspensions pooled from three mice.
3
Single-Cell RNA Sequencing of Murine Lung Cells
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After incubation with Percp-Cy5.5 anti-mouse CD45 antibody (BD) and Ghost Dye™ Red 780 (Tonbo), CD45+ cells from lung cell suspensions pooled from three mice were sorted on a BD FACS ARIA II cell sorter.
To ensure the quality of samples, automated cell counting and sample quality tests were performed (Countess® II Automated Cell Counter with AO/PI reagent) after preparation of a single cell suspension. Single-cell libraries were constructed using Chromium Controller, Single Cell 5’ Library, and Gel Bead Kit (10x Genomics), and the quality of each library was assessed by Agilent 4200, then sequenced via the Illumina Novaseq platform. The Cell Ranger (version 3.0.2) was applied to demultiplex and align reads using mouse GRCm38/mm10 as a reference genome, and was also used to generate gene-barcode matrices. To combine data from all six samples, the aggr pipeline of Cell Ranger was employed.
To ensure the quality of samples, automated cell counting and sample quality tests were performed (Countess® II Automated Cell Counter with AO/PI reagent) after preparation of a single cell suspension. Single-cell libraries were constructed using Chromium Controller, Single Cell 5’ Library, and Gel Bead Kit (10x Genomics), and the quality of each library was assessed by Agilent 4200, then sequenced via the Illumina Novaseq platform. The Cell Ranger (version 3.0.2) was applied to demultiplex and align reads using mouse GRCm38/mm10 as a reference genome, and was also used to generate gene-barcode matrices. To combine data from all six samples, the aggr pipeline of Cell Ranger was employed.
4
Isolation and Characterization of Brain Cell Types
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Isolated brain cells were prepared from C57BL/6J mice. Tissue was digested by papain at 37 °C for 1 h (2 mg/mL, LS003119, Worthington, USA) in DMEM medium. Dispersed cells were filtrated with a nylon mesh (70 μm). The cells were resuspended in Percoll density gradient (30%, 17-0891-09, GE Healthcare, USA) and centrifuged (900×g) at 25 °C for 25 min. Next, the cells in the bottom were collected. After washing in PBS containing 2% FBS, the cells were blocked with FcR Blocking Reagent (130-092-575, Miltenyi Biotec). Astrocytes, microglial cells, neurons, and endothelial cells were marked by flow cytometry42 (link), 43 (link), 44 (link), 45 (link). Cells were stained with PE anti-mouse ACSA-2 (130-116-244, Miltenyi Biotec, Germany), FITC anti-mouse/human CD11b antibody (101205, BioLegend, USA), PerCp-cy5.5 anti-mouse CD45 antibody (561869, BD Pharmingen, USA), APC anti-mouse NCAM-1/CD56 allophycocyanin MAb (FAB7820A-100, R&D, USA), and Brilliant Violet 605™ anti-mouse CD31 (102427, BioLegend, USA). After staining, the samples were sorted by FACSAria II SORP (BD Biosciences, USA), and the data were analyzed using FlowJo_V10 (FlowJo). Samples were gated for ACSA-2+ (astrocytes), ACSA-2–CD11b+CD45dim (microglial cells), NCAM-1/CD56+ (neurons), and CD31+ (endothelial cells). The RNeasy®-Micro Kit (74004, QIAGEN , Germany) was used for RNA extraction.
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