Cd3e alexafluor 647 clone 17a2

To induce immune tolerance, APP mice were injected at days 0, 2 and 10 with 7.5mg/kg IP with GK1.5 anti-CD4 monoclonal antibody (Bioxcell). To confirm CD4 T-cell depletion, blood was taken on day 12 by retro orbital sampling into heparin coated tubes. CD4+ T lymphocytes were quantified using FACS analysis on a BD Fortessa using standard protocols with CD45-FITC (clone 104 BD Pharmigen), CD3e-AlexaFluor 647 (clone 17A2, eBioscience) and CD4-PE (RM4-4 clone, BioLegend) antibodies. GK1.5 treated animals had reduced CD4 as evidenced by a ratio of CD4+ lymphocytes/ total CD3+ lymphocytes of 0.04 +/- 0.008 (mean +/-SEM) in the treated mice compared to 0.47 +/- 0.003 from untreated mice.

The anti-CD4 monoclonal antibody GK1.5 was produced from a hybridoma acquired from American Type Culture Collection, ATCC® TIB-207 [38 (link), 39 (link)]. For immunodepletion induction, 11- to 12-week-old APP751SL mice were treated at day 0 and day 2 by intraperitoneal injection of 150 μg GK1.5 while a control group (i.e., non-immunotolerized mice) was administered with PBS. On day 4, blood was taken by retro orbital sampling on a heparin-lithium coated tube (microvette®, Sarstedt AG, Germany). CD4+ T lymphocytes were quantified using FACS analysis on a FACSCantoII flow cytometer using standard protocols with CD45-FITC (clone 104 BD Pharmigen), CD3e-alexa Fluor 647 (clone 17A2, eBioscience), and CD4-PE (RM4–4 clone, BioLegend) antibodies. All GK1.5-treated animals displayed reduced CD4 as evidenced by a ratio of CD4+ lymphocytes/total CD3+ lymphocytes of 0.01 ± 0.01 in the treated group compared with 0.48 ± 0.20 in the untreated mice.