Hey! So if you’re getting super low colony counts in your HeLa invasion assay, especially for your wild-type Shigella, there are a few things you might wanna tweak. First, double-check your MOI—maybe try increasing the number of bacteria per HeLa cell a bit to boost initial invasion. Also, timing really matters: make sure your invasion time before gentamicin treatment is long enough for the bacteria to actually get in (usually 30–60 mins works, but you might need to test a bit). For the gentamicin step, be careful not to overdo it—high concentrations or too long an incubation can wipe out even the internalized bugs. Also, make sure your washing steps aren’t too harsh, ‘cause that can dislodge HeLa cells or mess with the internal bacteria. And finally, your plating dilution might just be off—so maybe plate a few different dilutions to catch the sweet spot. It can take a bit of fiddling, but you’ll get those 300–500 CFUs soon!