Table 2 Used antibodies for the flow cytometry
Tube | AB against | Name + Producer | Order-Number | Concentration/ Used Amount/ Dilution | Detected cells | Channel/ Laser |
|---|---|---|---|---|---|---|
1 | Untreated | ToProTM −3 Iodide, Life Technologies, Thermo Fisher Scientific, Carlsbad, USA | T3605 | dead cells | R1/Red 635 nm | |
2 | CD3 conjugated with FITC Clone CA17.2A12 | Mouse anti dog CD3 FITC; Bio-Rad, Hercules, USA | MCA1774F | 0,1 mg/ml 5 μl 11:1 | T-lymphocytes [25] | B1/Blue 488 nm |
2 | CD4 conjugated with PE Cy7 Clone YKIX302.9 | Anti-canine CD4 PE Cyanine 7; eBioscience, San Diego, USA | 25–5040-42 | 12 μg/ml 5 μl 11:1 | B2/Blue 488 nm | |
2 | CD11b conjugated with PerCP Vio® 700 Clone REA592 | CD11b PerCP-Vio® human and mouse; MACS Miltenyi Biotec, Bergisch Gladbach, Germany | 130–109-289 | 30 μg/ml 5 μl 11:1 | granulocytes, monocytes, NK-cells, subpopulation of T−/B-lymphocytes [19] | B2/Blue 488 nm |
2 | CD14 conjugated with PE Clone TÜK4 | Mouse anti Human CD14:RPE; Bio-Rad, Hercules, USA | MCA1568 PE | ca. 0,1 mg/ml 7,1 μl 8,04:1 | monocytes and macrophages, sporadic granulocytes [12] | B3/Blue 488 nm |
2 | CD45 conjugated with AF 647 Clone YKIX 716.13 | Rat anti Dog CD45 Alexa Fluor 647; SeroTec, Hercules, USA | MCA1042A647 | 0,05 mg/ml 5 μl 11:1 | all leucocytes in peripheral blood [6] | R1/Red 635 nm |
3 | Isotype CD 3 FITC Clone W3/25 | Mouse IgG1 negative control FITC; SeroTec, Hercules, USA | MCA928F | 0,1 mg/ml 5 μl 11:1 | B1/Blue 488 nm | |
3 | Isotype CD4 PE Cy7 Clone eBR2a | Rat IgG2a kappa Isotype Control PE Cyanine 7; eBioscience, San Diego, USA | 25–4321-82 | 0,2 mg/ml 5 μl 11:1 | B2/Blue 488 nm | |
3 | Isotype CD11b PerCP Vio® 700 Clone REA293 | REA control PerCP Vio 700 human; MACS Miltenyi Biotec, Bergisch Gladbach, Germany | 130–113-441 | 5 μl / 11:1 (new load: 1 μl / 51:1) | B2/Blue 488 nm | |
3 | Isotype CD14 PE | PE Mouse IgG2a, κ isotype Ctrl; BioLegend, San Diego, USA | 400,214 | 7,1 μl 8,04:1 | B3/Blue 488 nm | |
3 | Isotype CD45 AF 647 | RAT IgG2b negative Contral AlexaFluor 647; SeroTec, Hercules, USA | MCA1125A647 | 5 μl 11:1 | R1/Red 635 nm | |
4 | CD21 conjugated with PE Clone CA2.1D6 | Mouse anti canine CD21:RPE; Bio-Rad, Hercules, USA | MCA1781PE | 0,05 mg/ml 5 μl 11:1 | B-lymphocytes | B4/Blue 488 nm |
5 | Isotype CD21 PE Clone W3/25 | Mouse IgG1 negative Control:RPE; Bio-Rad Hercules, USA | MCA928 PE | 5 μl 11:1 | B4/Blue 488 nm |