Low protein binding microcentrifuge tube

Synthetic Aβ40 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-NH₂) was purchased from Proteogenix (Schiltigheim, France). Stock solutions were prepared by dissolving 1 mg of the peptide to a final concentration of 250 μM and adding 20 mM sodium phosphate buffer, 0.04% NH3, and NaOH to a final pH of 11. Then, the peptide was sonicated for ten minutes (Fisherbrand Pittsburgh, PA, USA, FB15051) without sweep mode and stored at −80 °C until needed [23 (link)].
All samples were prepared in low protein-binding microcentrifuge tubes (ThermoFisher Scientific, Waltham, MA, USA). The pH was measured at the start and end of all the aggregation reactions to confirm that it remained stable. For the pH change assays, 100 µL of sample after 24 h of aggregation at pH 7.4 was used, and 20 mM HCl was added to reach a pH of 5.5. For aggregates formed at pH 5.5, 20 mM NaOH was added to reach a pH of 7.4. For the seeding assays, 300 µL of preformed aggregates was obtained after 24 h of aggregation and 5 min of sonication. Preformed seeds were added to the corresponding aggregation reactions to a final seed concentration of 2.5 mM. The monomeric concentration of Aβ40 used was 25 µM. The control of unseeded reaction has the buffers at pH 7.4 and 25 µM of monomeric Aβ40 (everything but the seeds).

For each putative mutated neoepitope being investigated, HLA class I multimers were labelled with a unique combination of two different streptavidin conjugated fluorochromes, to enable the simultaneous detection of two-colour combinations of multiple mutated neoepitope multimers in a single sample [13 (link)]. The streptavidin conjugates used were PE [BD], BB515 [BD], BV421 [BD], and BV650 [BD], allowing for 6 different two-colour combinations. For staining, either 3 μl of BV421 or PECy7, 2 μl of APC, BB515 OR BV650, or 1 μl of PE multimers per sample were taken into low-protein binding microcentrifuge tubes [Thermo Fisher Scientific] and centrifuged at 9600 x g for 2 min at 4 °C. The supernatants were transferred to fresh low-protein binding microcentrifuge tubes and adjusted to a volume of 50 μl with 5% HS, PBS. PBMCs were multimer stained as normal (Additional file 1), and also stained with anti-CD8-APC (1:50) [BD] and live/dead dye (1:200) [Life Technologies] for 30 min at 4 °C. PBMCs were then washed twice in 5% HS, PBS and resuspended in 200 μl of buffer for acquisition using an LSRII [BD] and analysis using FACSDiva software [BD].

Hind paw leukocytes were isolated by crushing the tissue using mortar and pestle in PBS with 2% FCS. The resulting suspension was filtered over the cell strainer, followed by centrifugation (500 x g, 5 min, 2°C) and erythrocyte lysis in ACK buffer (150 mM NH₄Cl, 0.1 mM EDTA (disodium salt), 1 mM KHCO₃). Bone marrow cells were isolated by flushing femurs (cut at extremities) with PBS supplemented with 2% FCS, followed by red blood cell lysis with ACK buffer. Neutrophils were isolated from bone marrow cells by negative selection using mouse Neutrophil Isolation Kit (Miltenyi Biotec, catalog # 130-097-658) and autoMACS Pro magnetic cell separator (Miltenyi Biotec) according to manufacturer’s instructions. For LPS activation, 2×10⁶ cells in 700 µL IMDM with 0.1% FCS were placed in low protein-binding microcentrifuge tubes (Thermo Fisher Scientific). Subsequently, the cells were activated with 10 ng/ml LPS (L4516, Sigma-Aldrich) for 3 hours at 37˚C, 5% CO₂. For pervanadate activation, pervanadate was prepared by mixing 10 mM sodium orthovanadate with 0.3% hydrogen peroxide followed by 20 min incubation at room temperature. 100 µl of the resulting mixture was used for activation of 1.2 × 10⁷ cells in 1 ml media (20 min at 37°C).