Cytokine Profile Analysis of Spinal Cord Injury

Cytokines profile of CM derived from control, SCI + V, SCI + CM after 15 days and 30 days for the segment R1 was performed by using a Rat Cytokine Array Panel A according to the manufacturer’s instructions (R&D Systems, Inc., Abingdon, UK). Conditioned media from rostral segments from spinal cord tissue (all experimental groups and control) were obtained according to our previous study and afterwards processed for Cytokine profile detection [25 (link)]. We have used a similar procedure as in our previous study [26 (link)]. Briefly, the array membranes were first incubated in the blocking buffer for 1 h. Afterwards 200 µL of CM were mixed with the detection antibody mixture and incubated for 1 h at room temperature. After removing the blocking buffer, the sample/antibody mixture was added to array membranes and incubated overnight at 4 °C. Next day, the membranes were washed with the buffer and then incubated with Streptavidin-HRP solution for 30 min at room temperature. The membranes were finally washed with buffer 4 times and the bound antibodies were detected by chemoluminescence using a chemireagent mix. The membranes were quantified by densitometry using ImageJ software. Background staining and spot size were analyzed as recommended by the manufacturer. Normalization was done with control expression.