Chemokine Quantification using CBA

Chemokine quantification was performed using CBA – Human Chemokine Kit (BD Biosciences, USA), following the manufacturer’s instructions in culture supernatants. Briefly, 50 µl of capture beads for MCP1/CCL2, RANTES/CCL5, IL-8/CXCL8, MIG/CXCL9, and IP10/CXCL10, 50 µl of Detection Reagent and 50 µl of the studied sample or standard were added consecutively to each sample tube and incubated for 3 h at room temperature, in the dark. Next, the samples were washed with 1 ml of wash buffer and centrifuged. After discarding the supernatant, the pellet was resuspended in 300 µl buffer and analyzed in a FACS LSR Fortessa flow cytometer (BD Biosciences, USA) (Souza et al., 2021 (link)). Raw data were then analyzed using FCAP Array software (BD Biosciences, USA). The detection limits of each chemokine were as follows: 2.7 pg/mL for MCP1/CCL2, 1.0 pg/mL for RANTES/CCL5, 0.2 pg/mL for IL-8/CXCL8, 2.5 pg/mL for MIG/CXCL9 and 2.8 pg/mL for IP10/CXCL10.