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6 protocols using cd19 pecy5

1

Multiparametric Analysis of Immune Cells and Brain Pathology

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Splenocytes were isolated from spleens for flow cytometric analysis. Antibodies against the following surface markers were provided by: CD3 FITC (eBioscience™), CD4 PE (eBioscience™), CD8a Alexa Fluor 700 (eBioscience™), CD161 APC (eBioscience™) and CD19 PE-Cy5 (eBioscience™). Dead cells were stained by LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Life Technology). For immunohistochemical brain analyses, the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde in 0.1 M PBS for 2 days. Brains were cryoprotected by incubation in a 30% sucrose/0.1 M PBS solution. Sagittal brain sections were cut on a freezing microtome (Leica) and collected serially. Immunohistochemistry was performed on free-floating microtome-cut Sects. (10 μm in thickness). Sections were incubated with different antibodies: anti-mouse Iba1 (Polyclonal, 1:1000 Wako), anti-mouse CD68 (Clone FA-11, 1:200; BioRad), β-Amyloid (Clone 6E10, 1:1000; BioLegend), β-Amyloid 1–42 (polyclonal, 1:100; Millipore), anti-mouse GFAP (Clone GA-5, 1:100, Novus Biological) anti-human CD3 (Clone: CD3-12, 1:100, abcam) and anti-human Foxp3 (Clone 236A/E7, 1:100, Invitrogen). Appropriate secondary antibodies (Alexa Fluor 488, 594, or 647; Invitrogen) were used followed by incubation with DAPI.
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2

Characterization of Airway Immune Cells

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Immune cells were harvested from the airways via bronchoalveolar lavage (BAL). Briefly, mice were cannulated via a small tracheal incision and the lungs were flushed with 1 mL of sterile PBS. The collected BAL fluids were centrifuged at 1,500 rpm for 5 min at 4°C, and total cells were prepared for flow cytometry staining to determine the number and types of cells. Fc-blocked (1 μg/mL; eBiosciences) BALF cells were stained with anti-mouse SiglecF-PE (0.3 μg/mL; BD Pharmingen), CD11c-APC (0.3 μg/mL; eBiosciences), CD11b-PerCP (0.3 μg/mL; BioLegend), CD19-PECy5 (0.8 μg/mL; eBiosciences), Ly6G-PECy7 (0.8 μg/mL; BioLegend), Ly6C-APC-Cy7 (0.8 μg/mL; BD Pharmingen), and TCRβ-Pacific Blue (0.3 μg/mL; BioLegend). All samples were analyzed on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed by using FlowJo software (Tree Star Inc.).
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3

Purification and Sorting of Hematopoietic Stem Cells

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Miltenyi MACS magnetic bead purification was used to enrich BM MNC for CD34+ cells. The CD34+ fraction was then stained with CD2-PE-Cy5 (eBioscience, clone RPA-2.10), CD3-PE-Cy5 (eBioscience, clone UCHT1), CD4-TC PE-Cy5 (Invitrogen, clone S3.5), CD7-TC PE-Cy5 (Invitrogen, clone CD7–6B7), CD8-TC PE-Cy5 (Invitrogen, clone 3B5), CD10-PE-Cy5 (eBioscience, clone eBioCB-CALLA), CD11b-PE-Cy5 (eBioscience, clone ICRF44), CD14-TC PE-Cy5 (Life Technologies, clone TuK4), CD19-PE-Cy5 (eBioscience clone HIB19), CD20-PE-Cy5 (eBioscience, clone 2H7), CD56-TC (Invitrogen, clone MEM-188), Glycophorin A-PE-Cy5 (BD, clone GA-R2) and Thy1-biotin (eBioscience, clone 5E10, followed by CD34-APC (BD, clone 8G12), CD38-PE-Cy7 (eBioscience, clone HIT2), CD123-PE (BD, clone 9F5), streptavidin-APC-Cy7 (Life Technologies), CD45RA-FITC (Invitrogen, clone MEM-56) and DAPI. HSCe (DAPI-, Lin-, CD34+, CD38-) were cell sorted using either a BD FACSAria I with a 70 μm nozzle or a BD FACS SORP Aria-Ilu.
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4

BAL Immune Cell Profiling by Flow Cytometry

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Flow cytometry was used to determine the number and types of cells present in the BAL fluid. Fc-blocked (1 µg/ml; eBiosciences) BAL cells were stained with anti-mouse SiglecF-phycoerythrin (PE; 1 µg/ml; BD Pharmingen), CD45- allophycocyanin (APC; 1 µg/ml; eBiosciences), CD3-PECy5 (1 µg/ml; eBiosciences), CD19-PECy5 (1 µg/ml; eBiosciences), CD11c-PECy7 (1 µg/ml; eBiosciences), MHCII-APC-efluor780 (1 µg/ml; eBiosciences), CD11b-V450 (1 µg/ml; BD Pharmingen), Ly6C-FITC 1 µg/ml; BD Pharmingen) and Ly6G-AlexaFluor700 (1 µg/ml; BD Pharmingen). Fixable Viability Dye eFluor® 506 (eBiosciences) was added to exclude dead cells from the analysis.
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5

Multiparameter Flow Cytometry Panel

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APC: anti-Ly6G (clone: 1A8, Biolegend); APCeF780: CD8 (clone: 53–6.7, eBioscience), CD45 (clone: 30-F11, eBioscience), CD24 (clone: M1/69, eBioscience); BV421: CXCR4 (clone: L276F12, Biolegend); eF450: CD11b (clone: M1/70, Invitrogen); FITC: Ly6C (clone: HK1.4, Biolegend); PE: CXCR2 (clone: SA044G4, Biolegend), SiglecF (clone: E50–2440, BD bioscience); PECy5: CD19 (clone: 1D3, eBioscience); PECy7: CD4 (clone: GK1.5, eBioscience); PEeF610: CD11c (clone N418, eBioscience); PEVio770: CD64 (clone: REA286, Miltenyi Biotec); PerCPeF710: MHCII (clone: M5/114.15.2, eBioscience).
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6

Multiparameter Flow Cytometry Panel

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APC: anti-Ly6G (clone: 1A8, Biolegend); APCeF780: CD8 (clone: 53–6.7, eBioscience), CD45 (clone: 30-F11, eBioscience), CD24 (clone: M1/69, eBioscience); BV421: CXCR4 (clone: L276F12, Biolegend); eF450: CD11b (clone: M1/70, Invitrogen); FITC: Ly6C (clone: HK1.4, Biolegend); PE: CXCR2 (clone: SA044G4, Biolegend), SiglecF (clone: E50–2440, BD bioscience); PECy5: CD19 (clone: 1D3, eBioscience); PECy7: CD4 (clone: GK1.5, eBioscience); PEeF610: CD11c (clone N418, eBioscience); PEVio770: CD64 (clone: REA286, Miltenyi Biotec); PerCPeF710: MHCII (clone: M5/114.15.2, eBioscience).
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