Identification of cytokines present in CM. (A) CM was incubated with proteome profile array membrane for cytokine detection. BM was used a negative control to detect any noise or background. Localization of duplicate dots of IL6, IL8, and CCL2 on the membrane are indicated with arrows. (B) Supernatant was collected from CMI, n = 4 and CMD, n = 4, CLL PBMC were cultured in BM for 48 hours and the concentration of IL6, IL8, and CCL2 were determined by ELISA. Means are depicted as horizontal bars ± standard error bars, P values were calculated using Welch t test, ∗P < .05 indicates statistically significance. (C) CLL PBMCs (CMD and CMI) were cultured in CM either in presence of IL8-specific neutralizing antibody (left) or in presence of IL6-specific neutralizing antibody (middle) or in presence of CCL2-specific neutralizing antibody (right). Cell viabilities were measured after 48 hours (n = 5 for each group). Percent cell viability was calculated relative to day 0 (100%). Error bars represent standard error of the mean. P values were calculated using paired t test, and P value < .05 indicate statistical significance. (D) Cell viability of CM-dependent CLL measured PBMCs were cultured in BM with recombinant human cytokines (IL6, IL8, and CCL2) at 10 ng/mL or in combination with cytokine specific neutralizing antibody and isotype control (1 μg/mL) for 48 hours. Percent cell viability was relative to day 0 (100%) (E) Bh3 profile of primary CLL PBMCs from both group in response to BH3 peptides (BIM = 0.01μM; BAD = 0.03μM; MS1 = 2.5μM; HRK = 1μM) after exposing the cells to CM alone or CM with anti-CCL2 neutralizing antibody for 48 hours. Means are depicted as horizontal bars and error bars represent SEM. P values were calculated using 2-way analysis of variance, and P value < .05 indicate statistical significance. (F) Western blots from CMD CLL PBMCs treated with anti-CCL2 for 48 hours using the indicated antibodies.