Alexafluor 647 conjugated donkey anti goat antibody

Reagents for preparation of chemically defined medium (CDM) were purchased from VWR and Sigma. The medium was prepared as previously described (van de Rijn and Kessler, 1980 (link)). Todd-Hewitt broth, yeast extract, paraformaldehyde (PFA), nocodazole, Pitstop 2, Wortmannin, and Dynasore were purchased from Sigma. RPMI 1640 medium, Fetal bovine serum (FBS), Trypsin and other cell culture reagents were purchased from Hyclone, Thermo-Fisher. Gentamicin, AlexaFluor 647-conjugated donkey anti-goat antibody, AlexaFluor 568-conjugated phalloidin, AlexaFluor 488-conjugated transferrin or cholera toxin subunit B, and Fluorescein-conjugated dextran 70,000 MW were purchased from Invitrogen, Thermo-Fisher Scientific. Cytochalasin D was purchased from Calbiochem. Nystatin was purchased from Alfa Aesar. Anti-GAS antibody was purchased from Abcam.

Live H292 cells grown on a μ-slide 8 well glass bottom (Ibidi, Munich, Germany) and were infected with biofilm and planktonic bacteria of GFP-expressing GAS strains (ΔhasA or ΔhasAΔslo) using an MOI of 10. The media was removed, cells were washed in PBS (1X) and fixed by 4% PFA as described above. Extracellular bacteria were stained using a primary goat anti-GAS antibody (3.2 µg/ml) diluted 1:500 in 0.05% Bovine serum albumin (BSA) for 30 min. Excess staining was removed by washing in PBS (1X) three times. Secondary AlexaFluor 647 conjugated donkey anti-goat antibody (Invitrogen™, Thermo Fischer Scientific) diluted 1:500 in PBS (1X) was then added for 30 min in the dark. Excess antibody was removed by washing in PBS (1X) three times. Subsequently, cells were permeabilized using Triton X (0.01%) for 15 min at 37°C, washed, and stained using AlexaFluor 568 phalloidin (Invitrogen) using a 1:500 dilution in PBS (1X). DNA was stained by using Hoechst (Thermo Fischer Scientific) diluted to a working concentration of 1 µM in PBS for 30 min and washed for in PBS prior to imaging.