We verified the total protein content of the supernatants by Coomassie blue staining, which was similar for all strains (Figure. 1, for referee inspection only). The experiments were performed using different bacterial supernatant batches and results were remarkably consistent. All the supernatants were collected at the exact same bacterial culture’s optical density to harvest the bacteria in a comparable state between batches and strains. In our previous work we have shown that cholesterol-dependent cytolysins (SLO, PLY) displayed different kinetics and dynamics of their hemolytic activity compared to SLS. As a result, the total hemolytic activities of individual streptococcal supernatants were not represented by a simple sum of activities (concentrations) of their individual toxins but displayed more complex time- and amount-dependent behavior: e.g. – the activity of PLY/SLO was prevalent at the initial times of incubation and at relatively high amounts (volume) of supernatants, whereas SLS activity fully developed only after initial lag period but was prevalent when relatively low amounts of supernatants were used in the assays. The relative quantifications of toxins between streptococcal species and strains were performed in a previous publication [1] and are referred to throughout the manuscript. We added a paragraph in the result section (line 215-222) that summarizes those points. Moreover, in our current experiments we aim not only at the neutralization of the whole hemolytic secretome of streptococcus (as described in [1]) but also include (putative) cytotoxic/cytostatic activities that might be carried out either by hemolysins (SLO/SLS) or other not yet identified toxins that display cytotoxic/cytostatic but no hemolytic activities. For these reasons, in our current experiments, we use specific cytotoxic/cytostatic activities of total supernatants derived from a toxicity assay displayed in (Figure. 1), instead of concentrations of individual (partly unknown) toxins. However, we agree with the reviewers that using volume units is confusing. We therefore, edited our manuscript to display the lethal dose (LD%) interpolated from results shown in Figure 1 instead. Using LD% units accounts for the batch variability.
The authors have used bacterial culture supernatants to examine the cytotoxic effect on the THP-1, Jurkat, and Raji cell lines. Quantitation fo the the culture medium is required (by measuring the total protein content, or by providing some quantitative indicator). Volume of the culture supernatant is mentioned in the results. It is not quantitative, and will vary from batch to bath of the bacterial culture.