Ccf2 am loading kit

Following overnight culture, SE strains ΔfliC-pCX340 and ΔfliC-pCX340-galE were diluted into fresh LB medium with 12.5 μg/mL tetracycline and cultured at 37 °C for 3.5 h. IPTG was added to induce the expression of GalE protein. The cultured bacteria were washed twice with DMEM and transfected into HeLa cells with an MOI of 100, and then centrifuged at 1000 rpm for 10 min. Three hours post-infection, the HeLa cells were washed with DMEM four times and infected for another 4 h in DMEM. The cell monolayers were washed twice with DMEM and loaded in 300 μL DMEM supplement (60 μL 6 × CCF2/AM) with a final concentration of 1 μM. The 6 × CCF2/AM was freshly prepared with the CCF2/AM loading kit (Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37 °C for 2 h in the dark, and images were acquired using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany). The cells were excited at 405 nm, and emission at 450–470 nm and 520–540 nm resulted in either blue (cleaved) or green (uncleaved) fluorescence, respectively. Six hundred cells were randomly selected to calculate the percentage of GalE-positive cells.

NL4-3 and NL-AD8 BlaM-Vpr viral particles were produced in 293T as described above and in reference 61 (link). Target-activated CD4⁺ T cells were plated in 96-well plates at 10⁶ cells/mL and 100 μL/well. Viruses were added at the indicated concentrations, ranging from 10 to 500 ng/mL depending on the experiment, and cells were incubated for 2 h at 37°C. After extensive washing in PBS, T cells were loaded with the CCF2-AM loading kit (Invitrogen) in the presence of 1.8 mM probenecid (Sigma). Cells were incubated for 2 h at room temperature in the dark and then washed three times in PBS and fixed using 4% PFA. The cleaved CCF2-AM fluorescence (excitation at 405 nm, emission at 450 nm) was measured by flow cytometry on a FacsCanto II (BD). When indicated, target cells were cultivated with bacteria (50:1 ratio) for 24 h.

The TEM-1-based translocation assay is applicable for the evaluation of effector translocation using living host cells [21 (link)]. The nonfluorescent CCF2/AM substrate is converted to fluorescent CCF2 by the cellular esterases, and the resulting CCF2 emits a green fluorescence signal at 520 nm. The effector-mediated delivery of the TEM-1-fusion protein into the host cells induces the catalytic cleavage of the lactam ring of CCF2, leading to a detectable change of the CCF2 fluorescence from green to blue emission. A translocation assay was performed as described previously [22 (link)]. HeLa cells seeded at 4 x 10⁵ cells/well on coverslips in a 6-well plate with DMEM (Sigma) and 10% fetal calf serum were infected with precultured EPEC in DMEM at an MOI of 100. The cells were then centrifuged for 5 min and incubated at 37°C in an atmosphere of 5% CO₂. After 30 min, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the cultures at a final concentration of 1 mM, and the infection was allowed to proceed for an additional 1 h. The infected cells were then washed with PBS and stained with a CCF2/AM loading kit (Invitrogen). The cells were then observed with a fluorescence microscope equipped with a 4’,6’-diamidino-2-phenylindole (DAPI) filter set (365-nm excitation and 397-nm long-pass emission filters).