Tcs sp8 sted inverted fluorescence microscope

HD-11 cells were seeded on coverslips at a density of 1.5 × 10⁵ cells per well in 24-well plates and cultured overnight. After stimulation as indicated, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 5% FBS in TBSTx (10mM Tris-HCl,150mM NaCl, 20% Tween, and 0.1% Triton X-100) for 60 min. The cells were then incubated with anti-IpaJ antibody 4G6 (1:100) [32 (link)], followed by anti-mouse IgG (ab150113, Abcam, UK) and DAPI (D3571, Invitrogen, USA). The pYA3334-RFP plasmid was transformed into WT, ΔSPI-1, and ΔSPI-2 strains to express the red fluorescence protein. Images were acquired using a Leica TCS SP8 STED inverted fluorescence microscope (Leica, Germany).

IpaJ-TEM-1 fusions were constructed by cloning ipaJ into the TEM-1 fusion cloning vector, pCX340. The recombinant plasmid pCX340-ipaJ was then introduced into the WT, ΔSPI-1, and ΔSPI-2 strains, which were then used to infect HeLa cells. Cells infected with the WT-pCX340 strain was used as the negative control. After infection (3 h in DMEM supplemented with 1mM IPTG), cells were washed and loaded with CCF2-AM (Thermo Fisher, USA). Blue and green fluorescence were monitored using a Leica TCS SP8 STED inverted fluorescence microscope (Leica, Germany).