Normal mouse ig

Single cell suspensions were generated by mashing cervical, axillary, inguinal and mesenteric lymph nodes or spleens through a cell strainer (Falcon, Pittsburg, PA, USA). Single cell suspensions of splenocytes were then subjected to red cell lysis by hypoosmotic shock. Lymph node and red cell-lysed spleen cells were then resuspended in buffered salt solution (BSS) containing 0.1% (w/v) bovine serum albumin (BSA). Total lymph node or spleen cells were incubated with Pra1 (10 μg/ ml) or Aspf2 (10 μg/ ml) in PBS at 37°C for 30 or 45 min. For investigation of the influence of zinc on Pra1 binding, ZnCl₂ (1, 10, or 100 μM) was added while incubating cells together with Pra1. After washing bound Pra1 or Aspf2 were detected with a polyclonal anti-Pra1- (rabbit) or anti-Aspf2- (mouse) antiserum followed by PE anti-rabbit Ig polyclonal antibody (donkey; Dianova) or FITC anti-rabbit Ig polyclonal antibody (donkey; Dianova). For further stainings the samples were blocked with normal rabbit serum (1:500) or normal mouse Ig (20 μg/ ml, Sigma) followed by incubation with anti-CD4 mAb (Alexa Fluor 647) alone or together with anti-CD3 mAb (PerCp). For Kv1.3 detection, ShK-F6CA (0.3 μg/ml; Bachem AG, Bubendorf, Switzerland) was incubated together with mAb against cell surface proteins for 30 min at room temperature (Beeton et al., 2003 (link)).