Dmi6000 b with imc

For microscopic studies, 250–500 µg liposomes of DPPS were centrifuged at 10,000× g for 10 min at room temperature. The liposomal pellet was resuspended in Annexin binding buffer (ABB) and stained with Annexin V Alexa Fluor 350 conjugate according to the manufacturer’s protocol (Molecular Probes Inc., Eugene, OR, USA). PS exposure by Annexin V binding was visualized with a Leica DMI6000 B with IMC using a Leica DFC360 FX camera and AF 6000 software (Leica Microsystems, Vienna, Austria) (excitation wavelength 346 nm; emission wavelength 442 nm). For investigation of potential PS-peptide co-localization prior to PS-staining, incubation of 0.25–0.50 mM DPPS liposomes with 2 μM fluorescently labeled RDP22, ((5-6)-FAM-) RDP22 (excitation wavelength λ_ex = 495 nm; emission wavelength λ_em = 519 nm) for 10 min was performed.

In vitro toxicity spectroscopy studies were performed using Glomax Multi+ detection system (Promega, Madison, WI, USA). Micrographs were performed on a Leica DMI6000 B with IMC in connection with a Leica DFC360 FX camera and AF 6000 software (Leica Microsystems, Vienna, Austria).

TR146 cells were seeded in 8-well chamber slides (ibidi GmbH, Martinsried, Germany) with a seeding density of 2 × 10⁴ cells/300 µL and incubated for 2–3 days. Subsequently, medium was removed, (i) IL-1ß (i.e., 300, 400 and 800 ng/mL) and (ii) TNF-α (i.e., 200, 300 and 400 ng/mL) diluted in serum-free DMEM were added and incubated for 24 h. After 24 h, cells were washed twice with 1x Annexin binding buffer (ABB) of Vybrant Apoptosis Assay Kit #2 (Molecular Probes™, Thermo Fisher Scientific). Five microliters of Annexin V-Alexa Fluor 488 (staining phosphatidylserine (PS) exposing cells) and 2 µL propidium iodide (PI, staining DNA of necrotic cells) in 300 µL ABB were added and incubated at room temperature in the dark for 15 min. To remove unbound Annexin V-Alexa Fluor 488, cells were washed twice with ABB and covered with ABB. For imaging, a Leica DMI6000 B with IMC in connection with a Leica DFC360 FX camera and AF 6000 software was used. In addition to the brightfield transmission, the green fluorescence of Annexin V-Alexa Fluor 488 was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. For the red spectral region (PI), an excitation wavelength of 536 nm and an emission wavelength of 617 nm were used. Exposure time, intensity, and gain were fixed for all measurements.