Cedex bio analyzer

Concentrations of lactate, ammonium, glutamine, glutamate, and glucose were determined in single measurements with the Cedex Bio Analyzer (Roche, Switzerland). The measurement range was previously validated for each metabolite and samples were diluted accordingly when out of range. To determine the virus titer of the samples, two established assays were used. Firstly, the total influenza virus content was assessed based on a hemagglutination activity (HA) assay as described by Kalbfuss et al. [25 (link)]. Additionally, for the quantification of infectious particles, a 50% tissue culture infectious dose (TCID₅₀) assay using the influenza strain specific antibodies (Table 1) was performed as described by Genzel et al. The maximum standard deviation of the HA assay was ± 0.03 log₁₀(HAU/100 µL); the dilution error of the TCID₅₀ assay was ± 0.3 log₁₀ [26 (link)]. The concentration of total virus particles C_vir in the supernatant and the cell‐specific virus yield (CSVY_TCID, CSVY_HA) was determined using the following equation derived from [25 (link), 27 (link)]:
$$CSV{Y}_{TCID}=\frac{TCI{D}_{50,max}}{VC{C}_{max}}$$
$$C_{vir}=C_{ery}x{10}^{lo{g}_{10}\left(\frac{HAU}{100\mu L}\right)}$$
$$CSV{Y}_{HA}=\frac{C_{vir,max}}{VC{C}_{max}}$$ with VCC_max: maximum viable cell concentration obtained post infection, C_vir,max: maximum concentration of virus particles according to the HA assay, C_Ery: concentration of chicken erythrocytes used in the HA assay (2 × 10⁷ cells/mL).

C-LPNs were characterized with respect to morphology, elemental mapping, size, zeta potential, lipid coating, and antibody conjugation efficiency. The morphology of C-LPNs was visualized by transmission electron microscopy (TEM) using a JEM1010 system (JEOL, Tokyo, Japan). Prior to visualization, C-LPNs were briefly stained with a 1% uranyl acetate solution. Elemental mapping of carbon, oxygen, nitrogen and phosphorus present in C-LPNs was performed using energy dispersive X-ray spectroscopy-scanning transmission electron microscopy (EDS-STEM) using a JEM-2100F system (JEOL). Hydrodynamic size, size distribution, and zeta potentials were measured using dynamic light scattering and laser Doppler microelectrophoresis at an angle of 22° using an ELS8000 instrument (Photal, Osaka, Japan). The lipid content of nanoparticles was quantified by measuring phosphorus content using a phosphate assay¹⁸ (link). The content of immobilized antibody on nanoparticles was measured using a Cedex Bio Analyzer (Roche). The photothermal ability of LPNs was investigated by irradiating with a 808 nm laser using a diode laser beam (BWT Beijing Ltd., Beijing, China) at an output power of 1.5 W. Real-time temperatures were measured using an infrared camera (FLIR E60; FLIR Systems Inc., Danderyd, Sweden).

CALEC construct metabolic activity was measured by glucose consumption and lactate production pre- (0 hours) and post-hypothermic (24 hours) storage. CALEC construct media supernatant samples were analyzed for LDH release (LDH assay) and metabolic activity (glucose consumption and lactate production) using the CEDEX Bio Analyzer (Roche Diagnostics) as per the manufacturer’s instructions. The corneal media and HypoThermosol FRS contain about 5 and 4 nM glucose (and no lactate), respectively.

LDH activity is a well-established marker/indicator of cellular toxicity and lysis (32 (link)). LDH is a cytosolic enzyme that is released in the media only when the plasma membrane is damaged and is a surrogate marker for cell death. LDH was measured by a commercial colorimetric assay (Roche Diagnostics) on the CEDEX Bio Analyzer (Roche Diagnostics) and reported in a bar graph as the average LDH value ± SD.