LoFlo vs VLoFlo: Triplicate biological repeats (cultures) Cell counts made to 48 h Note: by 26 h cells round and shrink* in both LoFlo and VLoFlo By 47 h cell rounding and shrinkage pronounced and assay stopped * Noted previously in Dicty community that LoFlo is not ideal for long-term culturing. Typically, cells are grown only overnight in LoFlo, and transferred to fresh Lo Flo the following morning prior to imaging, so that LoFlo growth limited to <24 h but allowing HL-5-containing endosomes to disappear, reducing autofluorescence.