Prior to injection, 1 × 106 donor bone marrow cells from CD82KO (CD45.2) or WT (CD45.2) were treated with 5 μM EHop-016 (Selleckchem) or 50 μM NSC23766 (Cayman Chemical) in SFEM for 1 h at 37°C. Treated cells were then retroorbitally injected into BoyJ recipient mice (CD45.1). Recipient mice underwent total body irradiation 24 h prior to injection, which was administered as a single dose of 10 Gy. Mice were killed 16 h postinjection to assess chimerism of the bone marrow and peripheral blood. Blood and bone marrow samples were treated with Fc block prior to labeling with directly conjugated fluorescent antibodies FITC CD45.1 (A20; BD Pharmingen) and APC CD45.2 (104; BD Pharmingen) to assess chimerism. Samples were analyzed on the LSR Fortessa (BD Bioscience).
Fitc cd45.1 a20
Manufactured by BD
FITC CD45.1 (A20) is a monoclonal antibody conjugated with fluorescein isothiocyanate (FITC) that binds to the CD45.1 antigen expressed on certain cell types. It is commonly used in flow cytometry applications to identify and differentiate cells expressing the CD45.1 marker.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (4 mentions)
Cd45 apc, by BD (2 mentions)
Nsc23766, by Cayman Chemical (1 mentions)
Ehop 016, by Selleck Chemicals (1 mentions)
Percp cy5.5 cd3 145 2c11, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Facsaria 3 sorp, by BD (1 mentions)
Heparinized capillary tube, by Thermo Fisher Scientific (1 mentions)
Cd45.1 apc a20, by BioLegend (1 mentions)
Cd8 pecy7 53 6.7, by Cytek Biosciences (1 mentions)
B220 pe cy7, by Cytek Biosciences (1 mentions)
Cd4 pecy7 rm4 5, by BD (1 mentions)
5 protocols using fitc cd45.1 a20
1
Bone Marrow Cell Transplantation Chimerism
2
Competitive Bone Marrow Repopulation Assay
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For noncompetitive repopulation assay, 1 × 106 donor Lin– bone marrow cells from CD82KO or WT (CD45.2) were retroorbitally injected into recipient BoyJ mice (CD45.1). For a competitive repopulation assay, 1 × 106 donor Lin– bone marrow cells from CD82KO (CD45.2) were mixed at a ratio of 1:1 with Lin– BoyJ cells (CD45.1) and were retroorbitally injected into recipient chimeric mice (CD45.1/CD45.2). Recipient mice underwent total body irradiation, which was administered as a single dose of 10 Gy. Each month, blood was taken from tail snips to assess chimerism and immune cell differentiation. Cells were treated with Fc block prior to staining with the following directly conjugated fluorescent antibodies: FITC CD45.1 (A20; BD Pharmingen), APC CD45.2 (104; BD Pharmingen), PerCP-Cy5.5 CD3 (145-2C11; BD Pharmingen), BV421 B220 (RA3-6B2; Biolegend), Pe-Cy7 Ly6G (IA8; BD Pharmingen), and Pe-Cy7 CD11b (MI/70; BD Pharmingen). Labeled blood samples were analyzed using the LSR Fortessa (BD Bioscience).
3
Competitive Homing of CD82-Deficient Bone Marrow Cells
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For a noncompetitive homing experiment, 1 × 106 donor bone marrow cells from CD82KO or WT (CD45.2) were retroorbitally injected into recipient chimeric mice (CD45.1/CD45.2). For a competitive homing experiment, donor bone marrow cells from CD82KO were mixed at a ratio of 1:1 with BoyJ competition bone marrow cells and injected into chimeric recipient mice. Competitive homing experiments were also performed using isolated HSPCs from the bone marrow as described above. Recipient mice underwent total body irradiation, which was administered as a single dose of 10 Gy. Mice were killed 16 h postinjection to assess chimerism of the bone marrow and peripheral blood. Blood and bone marrow samples were treated with Fc block prior to labeling with directly conjugated fluorescent antibodies, FITC CD45.1 (A20; BD Pharmingen), and APC CD45.2 (104; BD Pharmingen) to assess chimerism. Samples were analyzed on the LSR Fortessa (BD Bioscience).
4
Immune Cell Profiling of Tumor and Lymph Nodes
5
Multicolor Flow Cytometry Protocol for Murine Immune Cell Analysis
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PB was collected from the retro-orbital plexus in heparinized capillary tubes (Fisherbrand, Pittsburgh, PA) and lysed in red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO). Cells were stained with the following antibodies: CD45.1-APC (A20) (Biolegend, San Diego, CA) or CD45.1-FITC (A20) (BD Biosciences, San Diego, CA), B220-PECy7 and CD8-PECy7 (53–6.7) (Tonbo Biosciences, San Diego, CA), CD45.2-V500 (104), B220- PerCPCy5.5 (RA3-6B2), Gr1-PerCPCy5.5 (RB6-8C5), Cd11b-PerCPCy5.5 (M1/70) and CD4-PECy7 (RM4-5) (BD Biosciences, San Diego, CA). All antibodies were used at 1:200 dilution. 4’,6-diamidino-2-phenylindole (DAPI) staining was used to gate live events. Analysis was performed on a LSR Fortessa and a BD FACSAria III SORP (Special Order Research Product, which contains the following LASERs: 405 nm, 445 nm, 488 nm, 562 nm, and 640 nm) (both BD Biosciences, San Diego, CA) and the data analyzed with FlowJo version 9.4.11 (Tree Star, Ashland, OR). To discern the four Confetti colors: the filter arrangements for each LASER were: CFP, 445 nm LASER—470/24 band-pass filter (BP); GFP, 488 nm LASER—515/20 BP and 505 long-pass filter (LP); YFP, 488 nm LASER—545/10 BP and 525 LP; RFP, 562 nm LASER—610/20 BP and 600 LP48 (link).
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