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T3SS RK13 cell Injection Assay

Seed RK13 cells

1. Remove the culture medium and wash 3 times with PBS follwed by trypsinization
2. Suspend RK13 cells with antibiotics free RPMI-10% FCS
3. Seed 2x10⁵/well RK13 cells on a 6 well plate or a glass bottom 3.5 cm dish 20 hrs before infection

Prepare E. coli culture

1. Grow E.coli-K12(SPI2/Signal-GFP/RFP), and E. coli K12(SPI2)in 4 mL of LB+0.4% arabinose with appropriate antibiotics at 37C overnight

10 hrs before injection

1. Centrifuge 4 mL of E. coli culture at 3,500 rpm for 10 min in the round tube.
2. Discard the sup and resuspend with 4 mL of MgM-MES(pH 7.2)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
3. 4 hrs later(5.5 hrs before injection) centrifuge the culture 3,500 rpm for 10 min
4. Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
5. 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup
6. Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube
7. Centrifuge the culture 5,000 rpm for 2 min and discard the sup
8. Repeat step 6 and 7 three times
9. Measure and adjust the concentration of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
10. Add 0.4% arabinose and appropriate antibiotics into this E. coli culture

Injection

1. Remove the RPMI on RK13
2. Add 1 mL of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
3. Incubate the plate at 37C, 5%CO₂ and observe the cells by Cofocal Laser Scannning Microscope(OLYMPUS FV-1000D) under blue and green exciter light at every 1.5 hrs after first exposure.