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.