Low tox h rabbit complement

14 of the 64 flow cytometry samples were screened by complement-dependent cytotoxicity. 7 had class II HLA sensitization and 7 lacked allosensitization. The CDC assay was performed as described by Diaz et al., with minor modifications(9 (link)). Briefly, Cells were added to each well with 1×10⁵ in 25 ul HBSS of Nunc^™ 96-Well Polypropylene MicroWell^™ Plate (Thermo Fisher Scientific, Waltham, MA), and incubated at 4°C for 30min with 25 ul/well of human sera treated with or without DTT (2.5mM in final) for 30min at 37°C. The cells were washed at 3 times with HBSS, then treated with 50ul/well of 11-fold dilution of Low-Tox®-H Rabbit Complement (Cedarlane, Burlington, NC) for 90min at 37°C. The cells were stained with FDA (0.5 ug/ml)/PI (2.5mg/ml, Sigma-Aldrich) at 4°C for 15min. Data were collected using a BD Accuri C6 Flow Cytometer and software (BD Accuri, Ann Arbor, MI, USA).