Cytokine/chemokine profile measurement by multiplex Enzyme-Linked ImmunoSorbent Assay (ELISA) array
Alterations in expression of major pro- and anti-inflammatory cytokines and chemokines in response to treatment with injection of bacteria was evaluated by multiplex ELISA kits (Cat No. MEM-004A, Qiagen). Briefly, peripheral blood of tumor bearing mice receiving PBS and 108 cfu/g bacteria bearing construct were collected in endotoxin-free EDTA containing collection tubes (BD Vacutainer Plus, US) immediately after completion of treatment period. Plasma was then collected by centrifugation of blood samples at 2500 × G for 10 min and kept at -80°C until further use. For analysis, 50 µL of thawed samples was added to each well of their respective row. The ELISA procedure was continued according to the manufacturer’s instructions and at the end, absorbance of the 96-well plate was read at 450 nm and 570 nm utilizing a plate reader. For correction of autofluoresence, the 570 nm absorbance was subtracted from final absorbance readings. Expression of each cytokine and chemokine was reported as the fold increase above PBS receiving group levels.