Glycine

100 μg of recombinant Tau 2N4R were incubated for 2 h at RT in 100 mM sodium citrate pH 6 (Sigma, cat. no. W302600), 100 mM sodium cyanoborohydride (Sigma, cat. no. 156159) and 20 mM formaldehyde (Fisher Scientific, cat. no. 28908) with a final volume of 100 μl. 10 μl of 550 mM glycine (Roth, cat. no. 3187) were added, and Zeba™ Spin desalting columns (Thermo Fisher, cat. no. 87767) were used according to manufacturer’s instructions to remove excessive sodium cyanoborohydride.

For immunofluorescence staining, cells were washed twice with phosphate-buffered-saline (PBS) without Ca²⁺/Mg²⁺ (LifeTechnologies, Carlsbad, CA, USA) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Paraformaldehyde was aspirated and cells were washed three times with PBS. Fixed cells were first permeabilized for 10 min in 0.2% Triton X solution and subsequently incubated for 1 h at room temperature in blocking solution (1% bovine serum albumin, 5% donkey serum, 0.3M glycine (Carl Roth GmBH & Co. KG, Germany) and 0.02% Triton X-100 (Thermo Fisher Scientific, Waltham, MA, USA) in PBS). Following blocking, primary antibodies were diluted in blocking solution and cells were incubated with primary antibody solution for 12 h at 4 °C. The following primary antibodies were used: chicken anti-SMI-32 (1:10.000, Covance, Princeton, NJ, USA), mouse anti-FUS (1:5000, Sigma Aldrich, St. Louis, MO, USA), rabbit anti-beta-III-Tubulin (1:3000, Covance, Princeton, NJ, USA), mouse anti-GM-130 (1:200, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-SOD1 (1:100, Cell Applications Inc., San Diego, CA, USA), rabbit anti-TDP-43 (1:400, Abcam, Cambridge, UK), rabbit anti-GFAP (1:1000 Chemicon, Temecula, CA, USA), mouse anti-vimentin (1:100, Sigma Aldrich, St. Louis, MO, USA). The nuclei were counterstained using Hoechst 33342 (LifeTechnologies, Carlsbad, CA, USA).

The feasibility to adhere to blood coagulation related stimuli and to accordingly change morphology was assessed with a method designed according to a protocol used by Cho et al.^{46 (link)}. 8 well PCA chamber slides (Sarstedt, Nümbrecht, Germany) were coated over night at 4 °C with 100 µg/ml fibrinogen (Sigma-Aldrich). Platelets were transferred to the coated wells and incubated in the presence of 1 mM ADP and 1 U/ml thrombin (both Sigma-Aldrich) for 90 min at 37 °C. After washing three times with PBS, the PLTs were fixated with Cytofix (BD Biosciences) for 10 min at 37 °C and washed twice with 100 mM glycine (Carl Roth, Karlsruhe, Germany) and once with PBS. The PLTs were permeabilized for 3 min with 0.2% Triton-X-100 (Sigma-Aldrich) at RT, washed three times with PBS and subsequently stained for 20 min at 37 °C with phalloidin-labelled in TexasRed (Invitrogen) diluted 1/70. The cells were washed three times with PBS and once with water. The grid of the PCA slide was detached, mounting solution with DAPI (Dianova, Hamburg, Germany) was applied and the slide was covered with a cover slip. Adhesion of PLTs to fibrinogen was analyzed by fluorescence microscopy using an Olympus IX81 microscope (Olympus) combined with a digital B/W camera (Olympus) and analyzed with Xcellence Pro image software (Olympus).

Alanine, aspartic acid, cysteine, glutamic acid, histidine, leucine, lysine, proline, threonine, tryptophan, valine, salicylic acid, pyridine, methanol, chloroform, methyl chloroformate (MCF), sodium bicarbonate, sodium sulfate, methoxyamine, ribitol, myo-inositol, xylose, pinitol, and iso-leucine were purchased from Sigma-Aldrich Chemie GmbH (Munich, Germany). Arginine and phenylAlanine were purchased from SERVA Electrophoresis GmbH (Heidelberg, Germany). Glycine, methionine, serine, malic acid, caffeic acid, succinic acid, arabinose, and saccharose from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Asparagine, mannitol, glucose, and galactose from Merck KGaA (Darmstadt, Germany). Sorbitol and glutamine from AppliChem GmbH (Darmstadt, Germany). MSTFA from Macherey-Nagel GmbH & Co. KG (Düren, Germany). Citric acid was purchased from Acros Organics (Thermo Fisher Scientific, Geel, Belgium).

Fibroblasts or transfected HEK 293 T cells were seeded into 6-well plates and treated with recombinant AGA (10–50 ng/ml) or 10 mM betaine (Sigma-Aldrich, Taufkirchen, Germany), glycine (Roth, Karlsruhe, Germany) or aspartic acid (Roth) for 48 h. All PCT substances were prepared as 1 M stock solutions in water. Cells were lysed as described above, and the lysates were used for enzyme activity measurements. For treating recombinant proteins, 5 μl of protein eluate was mixed with 40 μl folding buffer (100 mM NaCl, 0.1% Triton X-100, pH 4.5) and 5 μl 1 M substance and incubated for 3 h at 4 °C.

12 hours post-infection (hpi), amoeba infected with TIMER Lp strains were fixed in 4% Paraformaldehyde (Sigma Aldrich) for 20 min. Amoeba were then collected, washed in PBS and quenched in 0.1 M glycine (Roth) for 20 min. Finally, samples were resuspended in 1 ml PBS and kept at 4°C until analysis by flow cytometry. At least 20 000 events were acquired with the ImageStream X Mark II (Amnis) imaging flow cytometer. 5100 infected amoebae were identified and visualised using the IDEAS 6.2 software. Aspect ratio vs area was used to gate single amoeba, while intracellular bacteria were identified using green (Ex 488 nm, Em 528/65nm) and red fluorescence (Ex 561 nm, Em 610/30 nm) parameters. Images were acquired at 60x magnification using extended depth of field (EDF) and show brightfield, green fluorescence and red fluorescence channels as well as composite images of all three channels.

Poly(allylamine hydrochloride) (PAH), polystyrene sulfonate (PSS) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany), N-Hydroxysuccinimid (NHS), glacial acetic acid, sodium chloride (NaCl), sodium hydroxide (NaOH), glycine, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid-hydrochlorid (EDC), ethanol, HCl, 3-triethoxysilylpropylamine (APTES), 10 × PBS Buffer and BSA were purchased from Carl Roth GmbH (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Sodium acetate was purchased from Merck KGaA (Darmstadt, Germany). Human C-reactive protein, anti-hCRP-capture-antibody biotinylated (α-hCRPcb) and non-biotinylated (α-hCRPc) and anti-hCRP-detector-antibody (α-hCRPd) was purchased from Senova GmbH (Weimar, Germany). All proteins were used as received without further purification and all used antibodies are monoclonal. Sodium acetate buffer (NaAc) was prepared by dissolving Sodium acetate close to 10 mM in ultrapure water, pH was adjusted with HCl/NaOH to 4, 4.5 or 5 and filled up with ultrapure water to a final concentration of 10 mM NaAc. glycine–HCl Buffer (Gly-HCl) was pre-pared similarly but with glycine and adjusted to pH 2.5.