Effect of live bacteria on A549 cells attachment (2 hours incubation) in a 96 well plates

Purpose: to see the effect of bacteria of the attachment of A549 alveolar epithelial cells.
bacterial strains: Stenotrophomonas maltophilia  BAA84(D457), BAA2423(K279a), ATTC 13270
E coli clones cosmid control , pDR1, pDR2. EPI 100
bacteria grow over night in LB broth and then brought toan OD600 of 0.215 in PBS and then diluted in RPMI medium to approximately 5 x 10^5CFU/ml.
A549 cells were counted and seeded into 96 well plate in RPMI media with 5% FBS, cells seeded in 2.5 x 10^4 cell /ml(using 100 ul per well).After 48h remove the media and incubate the cells with 100 ul of bacteria and 100 ul of RPMI media for 2 h in 37 c ,5% CO2 incubator.
A group of cells was treated with trypsin 1% as fallowing:

• remove the media
• wash wells with 100 ul hanks solution 
• add 50 ul trypsin 1% for 90 seconds.
• remove trypsin and wait for 5 minutes
• wash 2-3 times with RPMI media 
• add 200 ul of RPMI media for each well. 

Another groups of cells were: washed thoroughly with hanks solution , cells without any washing and wells with no A549 cells.     

Result:
 MTT assay used to read the result of the cells attachment.