Preparing bacterial supernatant to be used on A549 epithelial cell culture

Purpose : to assets the effect of bacteria supernatant from different strains and clones of Stenotrophomonas maltophilia and E.coli  on the rounding and attachment of A549 epithelial cells.
S. maltophilia strains and clones were:
BAA84(D457)
BAA2423(K279a)

ATTC 13270
Grow in LB broth over night.
cosmid control , pDR1 pDR2,.....pDR10 , all grow in LB broth with chloramphenicol 20mg/ml

E.coli :DH5 alpha EPI 100 , grow in LB broth.

• remove LB by certerfugation.
• re suspend in PBS , brought to OD600=0.215 then dilute in RPMI medium to approximately 5 x 10^5 CFU/ml 
• after 24h static incubation 37 c in 5% CO2 cell free superntants were obtain by filtering the growth using 0.2 filter.
• supernatant can be used of kept at -20 c freezer.