Effect of live bacteria and the supernatant on A549 cells attachment after 24h incubation

Same procedure in experiments on 14 and 15 July 2014 with 24 h incubation time.

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.

Effect of bacterial supernatant on A549 cells attachment (2 hours incubation)

Purpose: to see the effect of bacterial supernatan 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.
The bacterial supernatant were prepared as in experiment in 11/7/2014.
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 bacterial supernatant 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.     

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

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.

Seeding A549 cells into 96 wells plate.

Purpose: to seed approximately equal number of cells in each well of the 96 well plate and carryout the desire treatment.

• Cells were grown in 25 cm3 flask,remove media 
•  wash the cells with PBS or HBSS, add 2 ml of 1% trypsin for 90 second to make cells detached from the plastic and remove the trypsin 
•  monitor the cell rounding under the microscope for 5-7 minutes during that time tap the flask against your palm to expedite cell detachment.
• add 5-10 ml of media, thoroughly pipette cells up and down, transfer a 8 ml of the cell suspension to sterile tube for counting 
•  do a 1:10 dilution with vital stain as fallowing 50 ul cells : 400ul HBSS : 50ul trypan blue.
• use 10 ul of above cell dilution with vital stain for cell counting using hemocytometer 

cells/ml = (aver number of cells per large square )10000 * dilution factor 

total cells in tube = cell/ml * volume of cell suspension.

A 25000 cells per well were seeded in each well. 

Incubate the cells in 37 C ,5%CO2 for 48 hours to reach 90-100% confluent.