F96 microwell plate

A total of 4,000 TZM-bl cells were seeded per well in 96-well plates and incubated overnight. One hundred microliters of the virus-containing supernatant was added to the cells, and the plates were incubated for 48 h. For the luciferase assay, 50 µl of lysis juice (p.j.k.) was added after washing the plates with PBS, and the plates were shaken for 15 min at room temperature. Next, the plates were frozen for at least 1.5 h at −80°C before being thawed. Lysates were resuspended and transferred to a white F96 Microwell plate (Nunc) for luminescent readout. One hundred microliters of beetle juice (p.j.k.) was added per well, and luminescence was measured with the Spark^® Microplate Reader (Tecan) at an integration time of 2 s. For the X-Gal staining, cells were washed with PBS and fixed in 0.06% glutaraldehyde (Sigma) and 0.9% formaldehyde (Sigma) for 10 min at 4°C. Cells were washed twice with PBS and staining solution was added containing 400 mM K₃[Fe(CN)₆], 400 mM K₄[Fe(CN)₆], 100 mM MgCl₂, and 20 mg/ml X-Gal. Cells were incubated overnight at 37°C and overlaid with 50% glycerol. Readout was performed optically with light microscopy.

A reporter cell line of human retinal pigment epithelial (RPE) cells, stably transfected with a plasmid containing the firefly luciferase reporter gene under the control of the IFN-stimulated response element (ISRE), was used to determine the activity of the different IFNα subtypes (27 (link)). Cells were seeded at 1.5 × 10⁵ cells per well in 12-well plates and incubated overnight. The next day, cells were stimulated with 10 ng/ml of the respective IFNα subtype for 5 h. Cells were then lysed with Passive lysis buffer (Promega) and frozen at −80°C overnight. After thawing, lysates were spun down and transferred to a white F96 Microwell plate (Nunc) before adding firefly luciferase substrate. Luminescent signal was measured using the GloMax^® Multi Detection System (Promega).

BMDMs were seeded in a Nunc™ F96 MicroWell™ plate (1 × 10⁵ cells/well) in differentiation medium and infected with C. albicans cells (1 × 10⁴ cells/well) in triplicates. Three hours after co-culturing, 4% Triton-X 100/PBS solution was added to lyse the macrophages. C. albicans cells were collected, plated, and colony forming units (CFUs) were counted. Data analysis and statistical analysis were performed in Graphpad Prism by using an ANOVA test with Bonferroni correction.

All tests for the interaction (i.e., co-aggregation) of GAPDH and Aβ peptides were performed under the following conditions: 30 μg/mL Aβ peptide, 30 μg/mL GAPDH, and 1 μg/mL tTG, dissolved in buffer containing 20 mM Tris HCl, pH 7.5, 20 mM NaCl, and 10 mM CaCl₂. Incubations were at 37°C for 18 h for electrophoresis or 48 h for the filter-trap assay and atomic force microscopy (AFM).
To analyze the interaction between GAPDH and biotinylated Aβ42, we developed a new ELISA-based test. Purified GAPDH was diluted to a concentration of 4 mg/mL in PBS and immobilized for 1 h on an F96 MicroWell™ plate (Nunc, Denmark). The plate was washed with PBS and blocked for 1 h with PBS containing 3% fetal calf serum. Immobilized GAPDH was then incubated for 1.5 h with 30 μg/mL Aβ42 dissolved in a buffer containing 20 mM Tris HCl, pH 7.5, 20 mM NaCl, and 10 mM CaCl₂. Factors affecting the interaction between GAPDH and Aβ42, such as transglutaminase (tTG) or cystamine, were added at this stage. For detection, avidin-peroxidase conjugate (Sigma-Aldrich, USA) was used. Visualization was performed using the tetramethylbenzidine protocol. The estimation of the interaction between GAPDH and non-biotinylated Aβ peptides was carried out using competitive inhibition of biotinylated Aβ42 binding to immobilized GAPDH. All experiments were performed in triplicate.

A total of 4,000 TZM-bl cells were seeded per well in 96-well plates and incubated overnight. The virus-containing supernatant was serially diluted 1:3 in culture medium and 200 µl of the diluted viral supernatant was added to the cells. Forty-eight post-infection, cells were washed with PBS and lysed using 120 µl of lysis juice (p.j.k.). Plates were incubated for 15 min before a freeze-and-thaw cycle was applied, to ensure complete lysis. Thirty microliters of homogenized lysates were transferred to a white F96 Microwell plate (Nunc) for luminescent readout. One hundred twenty microliters of beetle juice (p.j.k.) was added per well and luminescence was measured using the GloMax Discover (Promega) with an integration time of 5 s with one reading per well.

Growth of luminescent bacteria was measured as described previously [62 (link)]. Briefly, 1.0 × 10⁵luxABCDE-expressing M. marinum were plated on a non-treated, white F96 MicroWell plate (Nunc) containing 7H9-OADC medium and covered with a gas permeable moisture barrier seal (Bioconcept). Luminescence was measured for 30–70 h with 1 to 3 h intervals with a Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (Biotek). The temperature was kept constant at 32°C. For intracellular growth measurements, dilutions between 0.5 and 2.0 × 10⁵ of infected cells were plated as described above with HL5c medium containing 10 μM of amikacin to avoid extracellular growth of mycobacteria. Luminescence was also measured for 30–70 h with 1 h intervals at a constant temperature of 25°C.