CD99L2 supports aggregation of CD99L2-transfected CHO cells. (A) Mock-transfected CHO cells (CHO) or CHO cells stably transfected with CD99L2_s (CHO-CD99L2_S) or CD99L2_L (CHO-CD99L2_L) were allowed to aggregate in the presence (+) or in the absence (−) of Ca⁺²/Mg²⁺ as described under “Materials and methods.” ***, P < .001 CHO-CD99L2s with Mg²⁺/Ca²⁺ versus CHO-CD99L2s without Mg²⁺/Ca²⁺; ***P < .001 CHO-CD99L2l with Mg²⁺/Ca²⁺ versus CHO-CD99L2l without Mg²⁺/Ca²⁺. (B) Mock-transfected CHO cells (CHO) or CHO cells transfected with CD99L2_L (CHO-CD99L2_L) or CD99 (CHO-CD99) were allowed to aggregate in the presence of 30 μg/mL of the following antibodies: control antibodies (co-IgG), affinity-purified antibodies against CD99L2 (anti-CD99L2), or CD99 (anti-CD99) as indicated. ***P < .001 CHO-CD99 + anti-CD99 versus CHO-CD99 + co-IgG. (C) Mock-transfected CHO cells (CHO) or CHO cells stably transfected with CD99L2_s (CHO-CD99L2_S; labeled with Cell Tracker Green) or CD99L2_L (CHO-CD99L2_L; labeled with Cell Tracker Green) or mixed populations of mock-transfected or transfected cells (as indicated) were allowed to aggregate. Cell aggregates were analyzed by immunofluorescence microscopy and the number of mock-transfected cells ■ or transfected cells ■ per aggregate were counted. Statistical analysis was done by Student t test. Each panel is representative of at least 3 experiments; each measurement was done in triplicate.