Invasion protocol

Invasion protocol

Seed 1 - 2x105 HEp-2 epithelial cells/well in 1 ml of DMEM 10% FBS (DF) in 24 well plate, spin for 10 min @ 500 rpm, incubate o/n @ 37C, 5% CO2. 3 wells/bacterial strain. Before starting the invasion the cells should be in a confluent monolayer!

Grow bacteria in 2 ml LB w/ appropriate AB w/o shaking o/n @ 37C. Lifetiter after growth will be 1 - 1.5x109/ml.

Dilute 100 ul of bacteria in 900 ul DF w/o vortexing, put on ice.

Put 24 well plates on ice for 2-5 min, then carefully suck off medium from HEp-2 w/ low "sucking rate", do not let cells dry!!

Immediately add 200 ul of bacterial suspension/well (= ca. 2x107 bugs/well = MOI of ca. 100). Spin down bacteria on cells @ 500 rpm, 4C for 10 min. Take a short look through the microscope at the cells.

Keep original bacterial suspension in DF on ice during the experiment for later plating.

Put in fridge (ca. 4C) for 30 min (allow bacteria to adhere but inhibit invasion).

37C, 5% CO2 for 60 min. During this time, prepare tubes for dilutions of cell lysates and plates.

Take a short look through the microscope and protocol the appearance of the cells (e.g. % detached from the bottom of the well, watch abnormalities like increased amounts of vacuoles; always compare cell infected w/ wildtype to those infected w/ mutants).

Wash cells 3x w/ 1 ml PBS @ RT. Do not let cells dry during washes!! (Carefully add PBS to one side of the well, suck it off on another side. Do sucking always @ same side of well. Here the cells will be sucked off of the bottom of the well.)

Add 1 ml of DF w/ gentamicin [100 mg/l]/well@ RT, incubate @ 37C, 5% CO2 for 15 min.

Wash cells 3x w/ 1 ml PBS @ RT.

Before the last wash, take a short look through the microscope. Check for large patches of detached or sucked off cells.

Lyse cells by adding 1 ml PBS 1% Triton X100 @ RT/well and vigorously pipetting, at least 20 times, up and down.

Dilute lysate in LB and plate different dilutions on LB plates. Plate each dilution in triplicate.

Dilute original bacterial culture (1:10 dilution in DF) and plate.

Under these conditions wt 14028 will invade w/ 10 - 20% of the inoculum, EE638, prgH w/ 0.1%. Therefore, plate 100 ul of 10-3 - 10-5 dilutions of wt suspension, undiluted - 10-2 of EE638 or prgH.

Count bacteria the next day.

Calculate invasiveness in % of inoculum.