Leukocytes were isolated from samples of canine brain tissue and peripheral blood using a previously established and published protocol20 –22 (link) and single cell suspension was created. Cells were stained with anti-canine CD3 (Serotec, NC0522562) conjugated with anti-mouse IgG DyLight 549 (BioRad, STAR117D549GA), anti-canine CD4 PE/Cy7 (eBioscience, 25-5040-42), anti-canine CD8a eFluor 450 (eBioscience, 48-5080-42), anti-mouse/rat FoxP3 APC (eBioscience, 17-5773-82), anti-human CD152-FITC (MyBioSource.com , MBS666569), antihuman PD-1 biotinylated antibody (R&D Systems, BAF1086) conjugated with streptavidin FITC (eBioscience, 11-4317-87), anti-canine CD45 eFluor 450 (eBioscience, 48-5450-42), anticanine CD11c (BioRad, MCA17785) conjugated with anti-mouse IgG FITC (BioRad, STAR117F), anti-human CD123 PE (eBioscience, 12-1239-41), anti-human CD83 APC (MACS, 130-098-889), and anti-human PD-L1 (CD274) PE/Cy7 (BD Bioscience, BDB558017). Cells were analyzed by Flowcytometry, using a BD LSRFOrtessa X-20 cell analyzer, and data was analyzed with FlowJo V.10.2 software.
Protocol detail
Canine Leukocyte Isolation and Phenotyping
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Anti cd3
Anti igg
Antibody
Blood
Brain
Canine
Cd123
Cd152
Cd274
Cell
Fitc
Flowcytometry
Human
Leukocytes
Mouse
Streptavidin
Tissue
Corresponding Organization : Indiana University – Purdue University Indianapolis
Other organizations : Indiana University Health, Purdue University West Lafayette, Center for Cancer Research
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Variable analysis
independent variables
- Isolation of leukocytes from canine brain tissue and peripheral blood
- Expression of CD3, CD4, CD8a, FoxP3, CD152, PD-1, CD45, CD11c, CD123, CD83, and PD-L1 on leukocytes
- Previously established and published protocol (citation 20-22) for isolating leukocytes
- No positive controls explicitly mentioned
- No negative controls explicitly mentioned
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