Albumin fraction 5

Recombinant FLAG-tagged YB-1 was produced in HEK293T cells using the pDream2.1/YB-1 expression vector (based on GenScript pDream 2.1, kindly provided by P.R. Mertens). Cells were lysed in 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100 in water supplemented with protease inhibitors (cOmplete, Roche; Basel, Switzerland). Recombinant YB-1 was purified by binding to anti-FLAG M2 affinity gel (Sigma; Taufkirchen, Germany) with subsequent FLAG-peptide mediated (100 µg/mL; ApexBio; Houston, TX, USA) competitive elution in TBS containing protease inhibitors. The eluate was concentrated, and FLAG peptide was depleted by filter centrifugation with Vivaspin 6 Filterfalcons (10 kda cut-off; Sartorius; Göttingen, Germany). Protein concentration was determined with the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific; Waltham, MA, USA).
In the functional assessment of rYB-1, equivalent concentrations of Albumin Fraction V (BSA, Roth; Karlsruhe, Germany) dissolved in TBS were used as a control for unspecific effects attributed to protein supplementation. To further confirm specificity of the observed effects, recombinant YB-1 was heat denatured or concomitantly applied with a YB-1 specific blocking antibody (sc-398340, Santa Cruz Biotechnology; Heidelberg, Germany) or its matched isotype control (mouse IgG2a κ light chain antibody).

Cells that were already stained for surface markers were fixed with 1.5% PFA (Sigma Aldrich, St. Louis, MO) for 10 min at room temperature. Intracellular staining was performed in FACS buffer (PBS, 0.5% albumin Fraction V (Roth, Karlsruhe), and 0.1% NaN₃) containing 0.1% saponin (Sigma Aldrich, St. Louis, MO). The cells were washed with the same buffer and resuspended in PBS for measurement.
For staining of transcription factors in differentiated T cells, surface marker staining was performed for 10 min on ice before the cells were fixed with True-Nuclear 1× Fix Concentration (BioLegend, San Diego, CA) for 1 h at room temperature. The T cells were permeabilized and stained for transcription factors (T-bet (4B10), BioLegend, San Diego, CA; GATA-3 (16E10A23), BioLegend, San Diego, CA; RORγt (Q21–559), BioLegend, San Diego, CA) in True-Nuclear 1× Perm Buffer (BioLegend, San Diego, CA) for 30 min at room temperature.

The degradation of the ADA-gelatin gel was determined using the Bradford Test, the ROTI® Nanoquant assay (Carl Roth, Karlsruhe, Germany). For this purpose, the TRIS buffer (pH 5 and pH 7) was completely exchanged weekly and replaced with new TRIS buffer. The collected samples were frozen at −20 °C. To determine protein concentration, a calibration curve was constructed using known bovine serum albumin (BSA) (Albumin Fraction V, Carl Roth, Karlsruhe, Germany) concentrations. The amount of protein was quantified using a UV-Vis spectrometer (Spectrostar nano, BMG LabTech, Ortenberg, Germany) at 590 nm and 450 nm.

To compensate possible differences in the accuracy of tissue dissection for the HPLC-ECD, HPLC-MS and the pyruvate quantification analysis, we additionally measured the protein content in the samples after Bradford (Fic et al., 2010 (link)) and normalized amine or cyclic nucleotide concentration to protein content. The pellet (see above) was resuspended in 120 µL (HPLC-ECD: DV, DL), 30 µL (HPLC-ECD: MMTG), or 500 µL (HPLC-MS: DV+ DL) 0.2 M NaOH. After an incubation (15 min, 0 °C), the insoluble material was sedimented (9391 g, 5 min). Finally, 2 µL (HPLC-ECD: DV, DL), 10 µL (HPLC-ECD: MMTG), or 2,5 µL (HPLC-MS: DV+ DL) of the supernatant were transferred into a final volume of 1 mL 1 x ROTINanoquant solution (Carl Roth, Karlsruhe, Germany). All samples and the external calibrator (1, 2, 3, 5, 10, 20 µg/mL Albumin Fraction V, Carl Roth, Karlsruhe, Germany) were analyzed with a plate reader (Infinite 200 Pro, Tecan, Männedorf, Switzerland).

Polarized neutrophils (500,000 cells/sample) were resuspended in FACS buffer [1 × PBS (Thermo Fisher) supplemented with 1% bovine serum albumin (Albumin fraction V, Roth), 1% human serum, and 0.01% sodium azide (Sigma Aldrich)] in a V-bottom plate. After washing once with FACS buffer, the cells were stained with Pacific Blue^TM-conjugated mAb to human CD95 [clone DX2, immunoglobulin G1 (IgG1), Biolegend, San Diego, CA], allophycocyanin (APC)-conjugated mAb to human CD54 (clone HA58, IgG1, Biolegend), APC-conjugated mAb to human CD182 (clone 5E8/CXCr2, IgG1, Biolegend), fluorescein isothiocyanate (FITC)-conjugated mAb to human CD66b (clone G10F5, IgM, BD, Heidelberg, Germany), APC-conjugated mAb to human CD62L (clone DREG-56, IgG1, Biolegend), and phycoerythrin (PE)-conjugated mAb to human CD11b (clone 2LPM19c, IgG1, Dako, Waldbronn, Germany) for 30 min at 4°C protected from light.
After two wash steps with FACS buffer at 4°C, the cells were resuspended in FACS buffer and measured with a BD FACS Canto II (BD) flow cytometer. Data analysis was conducted with FlowJo V10.0.7. Representative FACS plots showing gating strategy (Supplementary Figure S2A), percentage of cells or mean fluorescence intensity (MFI) are shown in Supplementary Figure S3 (CD66b, CD11b), Supplementary Figure S4 (CD62L), and Supplementary Figure S5 (CD95, CD54, CD182).

Immunofluorescence assays (IFA) of parasites for confocal and STED microscopy were performed as described previously in detail [83 (link)]. In brief, parasites were seeded to concanavalin A (Sigma-Aldrich) coated μSlide 8-Well glass bottom dishes (Ibidi), fixed the next day with 4% PFA (Electron Microscopy Sciences) in PBS (Gibco, Thermo Fisher Scientific) for 20 min at 37°C, treated with 0.1% Triton X-100 (Sigma-Aldrich) and 0.1 mg/ml NaBH₄ (Sigma-Aldrich) PBS solutions for permeabilization and quenching of free aldehyde groups. Blocking as well as incubation of primary and secondary antibodies (S3 Table) was performed with 3% w/v Albumin Fraction V (Roth) in PBS. Hoechst 3342 (Thermo Fisher Scientific) was added during secondary antibody incubation at a dilution of 1:1000. Unbound antibodies were washed off with 0.5% Tween-20 (Roth) in PBS.