Accucount blank particles

To evaluate the cytotoxicity of ASNase, vincristine (VCR), and daunorubicin (DNR), MTS (dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium) assays were performed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corporation, Wisconsin, USA) according to the manufacturer’s instructions and our previous publication [7 (link)]. Range of ASNase concentration used in the MTS assay was 0 - 4 IU/ml (5-fold dilutions), for VCR it was 0 - 50 nM (5-fold dilutions) and 0 - 3 μM (5-fold dilutions) for DNR. We seeded 1.2 × 10⁴ cells of leukemia cell lines and 1 × 10⁵-3 × 10⁵ patient cells. To evaluate the combined cytotoxicity of the 2 drugs, the number of live cells was determined by flow cytometry using DAPI (ThermoFisher Scientific Inc., MA, USA) and AccuCount Blank Particles (Spherotech Inc., IL, USA). The MTS assay was performed in at least 3 independent experiments. Cell counts were done in biological triplicates.

Cell growth was determined as the ratio of cells per defined volume of counting particles (2 μm AccuCount Blank Particles). For this, 79 μL of 50 µg/ml Tc and 500 μg/mL rifampicin (Sigma-Aldrich Chemie GmbH) in phosphate-buffered saline for transcription and translation inhibition, and 21 μL of AccuCount Blank Particles (Spherotech) for counting reference was used per sample. The cell solution was added in equal volumes (100 µL cell culture to 100 µL of inhibition and counting solution). Absolute cell counts per particle counts were determined by flow cytometry. For this, samples were measured for 2 min at 10 µL/min. The gating of the AccuCount particles and cells is shown in Supplementary Fig. 12 bottom. The reported data are from three biological replicates pooled from experiments performed on the same day. The doubling time of E. coli was measured to be 36.9 ± 1.2 min in our setup (Supplementary Fig. 13). We further determined that an inoculum of 1:20,000 of overnight culture into fresh media leads to an OD₆₀₀ of 0.034 ± 0.003 after 5 h incubation, therefore ensuring logarithmic growth throughout the experiments. Further, a calibration of optical density of a cell culture (OD₆₀₀) to our cell growth measurement using flow cytometry (counts/bead) was performed, which shows a linear correlation until an OD₆₀₀ of 0.2 (Supplementary Fig. 14).