Here, we describe two rapid non-radioactive and non-toxic ADCC and CDC assays allowing for analysis of samples collected from NB patients treated with anti-GD2 Ab ch14.18/CHO for the purpose of immune monitoring of ongoing clinical trials. Importantly, we demonstrated increased GD2 specific CDC and ADCC in three selected high-risk NB patients treated with ch14.18/CHO indicating expected effector functions of ch14.18/ CHO in treated patients. Among non-radioactive agents that can be used for such cytotoxicity assays, calcein-AM is one of the most prominent fluorescent dyes used for labeling of target cells. Here, expression of multidrug transporter P-glycoprotein is a hindering factor extruding calcein from target cells independently of CDC or ADCC. To avoid this problem, we selected LA-N-1 cells from a panel of NB cell lines. LA-N-1 showed low Pglycoprotein expression levels and subsequently low background signals after calcein labeling. Moreover, the most homogeneous and strongest GD2 expression and the highest level of GD2-specific lysis in contrast to all other NB cell lines of the available panel resulted in the decision to establish CDC and ADCC bioassays with LA-N-1 target cells. We also aimed to optimize the bioassays to reduce both preparation time required for effector cell isolation as well as sample volume, which is particularly important in a pediatric population. To this end, a minimum of 100 ml of serum and a blood sample Carbimazole containing 200,000 leukocytes are sufficient which can be isolated within 10 min from small blood volume of less than 500 ml. A second challenge to overcome is the fact that European NB trials are Butacaine generally multi-center in nature and require sample transfer to a central laboratory for analysis. Therefore, the impact of shipping conditions is of great importance. According to our present data, serum samples could be stored at RT for four days and subjected three times to repeated thawing and freezing without significant influence on CDC. For the analysis of ADCC, blood needs to be anticoagulated. Although EDTA is the most commonly used anticoagulant, it is known that it affects both erythrocytes and leukocytes, causing membrane damage.