Cd4fitc

Antibodies specific to bovine neutrophils (Clone#CH138A), CD25 (Clone#LCTB2A and Clone#CACT108A), and CD69 (Clone#KTSN7A), CD28 (Clone#TE1A) were obtained from the WSU Monoclonal Antibody Center (Pullman, WA). CD62L-FITC conjugate (Clone#CC32), CD4-FITC (Clone#CC8), CD8-PE (Clone#CC63), CD25-PE (Clone#IL-A111), CD28 (Clone#CC219), IL-10 Abs (Clone#CC320), anti-bovine IFNγ-PE (Clone#MCA1783) were procured from BioRad (Hercules, CA, USA), and all anti-mouse isotype secondary Abs were purchased from Biolegend (San Diego, CA, USA). Manufacturer-recommended concentrations for each Ab were used, typically 1.25–10 µg/mL in 100 µL reaction media.

Lymph node aspirates were collected into flow cytometry buffer and were analyzed immediately or stored at 4°C for a maximum of 24 hours. The flow buffer was composed of phosphate buffered saline (PBS) with 1% horse serum, 5mM ethylenediaminetetraacetic acid (EDTA) and 0.1% sodium azide. Flow cytometric immunophenotype of the neoplastic cells was determined using a FACScan instrument (BD Biosciences, Mississauga, Canada). Leukocytes were stained with a panel of 11 antibodies including: CD3_FITC (CA17.2A12, T lymphocytes), CD4_FITC (YKIX302.9, neutrophils and T-helper lymphocytes), CD5_FITC (YK1X322.3, T lymphocytes), CD8α_PE (YCATE55.9, cytotoxic T lymphocytes), CD14_PE (TUK4, monocytes), CD18_A647 (CA1.4E9, all leukocytes), CD21_PE (Ca2.1D6, B lymphocytes), CD22_PE (RFB4, Novus, B lymphocytes), CD34_PE (1H6, hematopoietic stem cells), CD45_FITC (YK1X716.13, all leukocytes) and MHCII (YK1X334.2, lymphocytes and monocytes) (Bio-Rad, Berkeley, CA, USA).

Blood was collected into EDTA tubes via cardiac bleeds and prepared using Dako Uti-Lyse erythrocyte lysing solution according to instructions. Blood was stained with AF647-labeled CSF1-Fc (6 (link)) and the following Abs: CD32^APC (Clone REA256, 1:40) and CD161^APC (Clone REA227, 1:40) from Miltenyi Biotec, B220^PE (Clone His24, 1:400; eBioscience), CD4^FITC (Clone W3/25, 1:10; Bio-Rad Laboratories), CD3^AF488 (Clone 1F4, 1:200), and SIRPα^PE (Clone OX-41, 1:400) from BioLegend. Myelin-depleted single-cell suspensions of brain were prepared as described in (21 (link)), except rats were perfused with physiological saline and heparin and then stained with CD45^EF450 (Clone OX1, 1:200; eBioscience) and CD11b/c^AF647 (Clone OX-42, 1:500; BioLegend). Peritoneal cavity cells were isolated as described in (22 (link)) and stained with SIRPα^PE and CD11b/c^AF488 as described above. Isotype controls were used for all flow cytometry data. Cells were analyzed by flow cytometry on a FACSCalibur, LSRFortessa X-20 or LSRFortessa (BD Biosciences). Analysis was performed with FlowJo software (FlowJo).