Citation from the protocol:
"Fixed samples microscopy
36-48 h before the experiment, detach a confluent TC7 cells monolayer with 0.25% trypsin/EDTA and distribute TC7 cells on coverslips no. 1.5 placed in wells of a 24-well plate in their normal growth medium. To follow entry events, seed 5 x 104 cells/well (approximately 50-75% confluence the day of the experiment). To favor cell-to-cell spread during longer infection times, seed 1 x 105 cells/well. (Optional) Fibronectin-coated coverslips can be used to permit faster attachment of the cells, but in this case, the number of cells seeded must be reduced about 25-50% to obtain confluence similar to what is described for non-coated coverslips.
The day before the experiment, a CR–positive colony harboring pTSAR1ud2.1 or pTSAR1Ud2.4s is picked from a CR-containing TCS ampicillin plate and used to inoculate one tube of 8 ml TCS broth supplemented with Amp and incubated overnight at 30 °C with shaking.
Notes:
CR induces T3SA activation and effector secretion; colonies with an active T3SA therefore display a red center where CR has accumulated while bacteria that have lost the virulence plasmid show up as larger white colonies (Figure 2).
Strains harboring pTSAR1Ud1.1 are used in place of pTSAR1Ud2.4 for multiple labeling experiments because it allows the use of the red laser/Cy3 filter for immunofluorescence."
In the second to last line of this paragraph, there is a typo; pTSAR1Ud1.1 should be read pTSAR1Ud2.1.
3/2/2015 12:40:36 AM Reply