Microtainer serum collection tube

VSG2-expressing GPI-PLC^−/− trypanosomes were pelleted from culture, washed with irradiation buffer (PBS, 154 mM glucose), and resuspended in irradiation buffer to a density of 10⁷ cells/ml. A volume of 1 ml of this resuspension was aliquoted into each well of a six-well tissue culture plate (Falcon 353046). Plates were UV-irradiated for 10 min in 1 min intervals using a FB-UVXL-1000 UV crosslinker (Fisher Scientific). Plates were swirled between 1 min intervals to ensure equal irradiation of trypanosomes. Irradiated cells were pelleted and resuspended in HMI-9 at a concentration of 15 × 10⁶ cells/ml. A volume of 100 µl of this solution (3 × 10⁶ trypanosomes) were injected i.p. into mice. This immunization procedure was performed twice on each mouse (day 0 and day 3). For live trypanosome comparison immunizations (Supplementary Fig. 3a), mice were infected on day 0 with 100 or 1000 live WT-VSG2 trypanosomes via i.p. injection. Infections were cleared with 250 ng berenil/mouse injected i.p. on the day trypanosomes were identified by blood smear (day 4 for 1000 injected, day 5 for 100 injected). Berenil treatment was repeated 24 h after initial treatment. Serum samples for ELISA analysis were obtained via submandibular bleed, and serum was separated from whole blood using Microtainer serum collection tubes (BD 365967).

Following anesthetization with isoflurane, serum was collected from mice via submandibular blood draw using 5.5 mm Goldenrod animal lancets. Serum BUN, creatinine and albumin were analyzed. Blood was collected using BD Microtainer serum collection tubes from the puncture site and then centrifuged at 1000 g for 10 min at 4°C to isolate serum; the serum was then shipped to IDEXX laboratories for analysis. Average lab values between 3 allele and control mice were compared with using a two-tailed t-test with unequal variance (total n = 44 3 allele mutants, 36 CTRL for phenotypic comparison).