Figure 1.
Picture and illustration of the set up.
(a) Photo of the acoustophoresis microfluidic system first presented by Augustsson et al. [27]. (b) Cross sectional view of cell distribution in the microchannel without ultrasound (left) and with ultrasound forming an ultrasound standing wave (right). (c) Illustration of acouscoustophoresis chip. For the present study, only one of the channel segments was used allowing cells to be exposed to ultrasound.
Figure 2.
Unaltered viability of BV2 microglial cell line following acoustophoretic processing (10Vpp and 20Vpp).
BV2 cells were passed through the acoustophoresis chip with the function generator set at 0, 10 and 20 Vpp. After going through the chip, the BV2 cells were seeded again for 24 and 48 h. Cell viability was measured by XTT (a), apoptotic nuclei appearance (b) and decrease of mitochondrial potential -Ψm- (c), showing no difference between experimental groups. Similar, no difference was detected by clonogenic assay (d) used to study survival and proliferation at 7 days following acoustophoretic processing. The graphs show the results from at least three separate experiments and the data are shown as means ± SD. Significance value P<0.05, ns denotes non-significant.
Figure 3.
Inflammatory response of BV2 cells upon LPS challenge following acoustophoresis is not changed.
After acoustophoretic processing, BV2 cells were seeded and stimulated the next day with LPS for 24 h. We observed no alteration due to acoustophoretic processing in the expression of iNOS (inducible nitric oxide synthase)(a,b), the release of proinflammatory cytokines IL-1β (χ), IL-12 (d), TNF-α (e) or the anti-inflammatory cytokine IL-10 (f). The graphs show the results from at least three separate experiments and the data are shown as means ± SD. Significance value P<0.05, ns denotes non-significant.
Figure 4.
Prostate cancer cell viability is not affected by acoustophoresis.
Cell viability was determined after acoustophoresis (0, 10 and 20 Vpp), for four prostate cancer cell lines: DU 145 (a), PC3 (b), LNCaP (c) and VCaP (d). Left panels show the percentage of cell death directly after acoustophoresis measured by trypan blue exclusion. Right panels show the cell death quantified at 24 and 48 h after acoustophoresis by flow cytometry. Cells negative for the DNA binding fluorochrome 7-Aminoactinmycin (7-AAD) were defined as viable. At least 10,000 cells were counted and the percentage of dead cells was determined. Cells not subjected to acoustophoresis were used as control cells. The graphs show the results from at least three separate experiments and the data are shown as means ± SD. Significance value P<0.05, ns denotes non-significant.
Figure 5.
Acoustic cell separation in a microchannel does not alter PSA secretion by prostate cancer cells.
The androgen receptor (AR) expressing cell lines LNCaP and VCaP were used to evaluate the impact of acoustophoresis on PSA secretion. After acoustophoresis run at 0, 10 and 20 Vpp, the secretion of PSA was measured in the absence or presence of the AR ligand R1881 (1 nM for 24 h) in the LNCaP cell line (a) and in the VCaP cell line (b). Cells not processed through the chip were used as control cells. The graphs show the results from three separate experiments and the data are shown as means ± SD. Significance value P<0.05, ns denotes non-significant.
Figure 6.
Mitochondrial respiratory function in human leukocytes and thrombocytes are not altered following acoustophoresis.
Leukocytes and thrombocytes were passed through the acoustophoresis chip run at 0, 10 and 20 Vpp. Maximal respiration during using both complex I- and complex II-linked substrates for thrombocytes (a) and leukotcytes (b) was unaltered after acoustophoresis, supporting that acoustophoresis does not affect metabolic pathways important for respiration. The remaining respiratory activity following inhibition of ATP synthase with oligomycin, so called Leak or state 4 respiration, was also unaffected by acoustophoresis, in thrombocytes (c) and leukocytes (d). This data confirm no effect of acoustophoresis on inner mitochondrial membrane integrity or changed utilization of proton motive force for other purposes than ADP phosphorylation. Unprocessed cells were used as control cells. The graphs show the results from three separate experiments and the data are shown as means ± SD. Significance value P<0.05, ns denotes non-significant.