Fig 1: Cytotoxicity of isolated aNK extracellular vesicles. Luciferase assay. EVs were isolated from 48 h conditioned medium of aNK cells as in Figure 1. ALL SupB15-fLuc cells (104 cells) or neuroblastoma CHLA255-fLuc cells (104 cells) were incubated in 96-well plates with aNK cells (104 cells; effector:target cell ratio 1:1) (columns 1 and 5) as the positive control, or different amounts of purified exosomes as indicated. After 6 h incubation, the luciferase substrate Beetle Luciferin (Promega, E1605) was added, and bioluminescence was quantified. Untreated samples were designated as 100% survival for CHLA255-fLuc or SupB15-fLuc (lanes 2 or 6, respectively) and results were expressed as per cent survival. fLuc: firefly luciferase labelled cells. *p < 0.05, **p < 0.01 (a) Acridine orange/propidium iodide fluorescence assay: 104 cells (SupB15 or CHLA-255) from log phase cell culture were transferred to 96-well dark plates in the presence (∎, solid line) or absence (△, dotted line) of EVs (40 μg) in a final volume of 100 μl. Similarly, 40 μg EVs isolated from neuroblastoma CHLA-255 cells was used as the negative control (⋄, solid line). The suspensions were incubated at 37°C, and the percentage of dead cells was calculated by the LUNA cell counter at various times (0, 6, 12, 24 h). (b) The acridine orange/propidium iodide fluorescence assay was performed using different cancer cell lines, as indicated. The percentage of dead cells, without (light box) or with EVs (40 µg) (grey box), was determined by the LUNA cell counter at 24 h. The error bars on the graphs are generated by the Excel software based on 4–8 readings in each test. *p < 0.05, **p < 0.01.
Fig 2: Analysis of A. niger MA317 transformants containing the Pgpd-mluc reporter construct using the pyrG** targeting method. A) Southern blot analysis. Genomic DNA was restricted with EcoRI or KpnI and size fractioned by electrophoresis on a 1.0 % agarose gel. For hybridisation, 32P-labelled pyrG probe (1255 bp, Figure 2A) or 3’ pyrG probe (684 bp, Figure 2A) were used. When digested with EcoRI and using the pyrG probe (upper panel), a signal of 9.0 kb corresponds with the wild-type pyrG locus, while a signal of 4.9 kb corresponds with integration of the Pgpd-mluc cassette at the pyrG locus. When digested with KpnI and using the 3’ pyrG probe (lower panel), a signal of 3.3 kb corresponds with the wild-type pyrG locus, while a signal of 4.8 kb corresponds with integration of the Pgpd-mluc cassette at the pyrG locus. Strains MA317.1 and MA317.3 have the wild-type pyrG locus, while strains MA317.2 and MA317.4-6 contain the Pgpd-mluc cassette at the pyrG locus. B) Lux activity assay. The lux activity assay described by Meyer et al. [12] has been slightly modified. Briefly, 100 μL of 2 x Minimal Medium [5] with 0.006 % yeast extract (w/v), 76 μL deionized water, 4 μL 25 mM luciferin (Promega, E1605) and 20 μL spore suspension (7.5*105 spores/mL) was pipetted together (in triplicate) in a well of a white, clear bottom, 96 wells plate (Greiner Bio-one, ref 655095) and incubated for 24 hours at 30 °C in the EnSpire Multiplate Reader (Perkin) with continuous measuring of lux and OD. Lux activities after 16 h of incubation are shown here. Strains MA317.1 and MA317.3 have no lux activity, while strains MA317.2 and MA317.4-6 show comparable lux activities.
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