Tuerk solution

Blood (50 mL) was taken using 10 mL S-monovets (Sarstedt, Nürnbrecht, Germany) each with 3.2% citrate, from HDs and HNSCC TPs before the onset of any therapeutic measures. “Healthy” is defined here as “no diagnosed tumor” at the time of blood collection. Additionally, tissue material from the primary tumor was collected. Donors’ monovets were centrifuged at 120 × g for 20 min. The exact number of platelets that were obtained has not been determined routinely. During the examination of the platelets under the microscope, random samples showed regular platelet counts, from 150,000/µL to 450,000/µL blood. The platelet-rich plasma was centrifuged at 360 × g for 20 min. The resulting pellet was resuspended in 400 μL to 500 μL RNAlater^TM (Qiagen, Hilden, Sweden). To check the purity of the platelets, 10 μL of platelet suspension was diluted with 90 μL of Tuerk solution (Merck, Darmstadt, Germany) and checked microscopically. The preparation was defined as pure if at most 5 nuclear-containing cells per 10 million platelets could be identified.

A time window ≤ 12 h after completion of sampling was allowed for sample processing. All laboratory analyses were performed by trained personnel unaware of clinical data. The CPDA-1-anticoagulated blood was used for cell counting and the preparation of blood smears. After red blood cell lysis and white blood cell (WBC) staining with Tuerk solution (Merck, Darmstadt, Germany), total WBC counts were determined by using a Fuchs Rosenthal hemocytometer. Thin blood smears were prepared from 5 μL of whole blood (glass slides: R. Langenbrinck, Emmendingen, Germany). Blood smears were stained by standard Pappenheim stain (Merck). Cell counts of neutrophils, lymphocytes, and monocytes were calculated by multiplying the percentages in Pappenheim-stained blood smears with the respective WBC counts.

To isolate peripheral blood mononuclear cells (PBMCs), fresh porcine, chicken, and bovine whole blood was obtained from an abattoir in EDTA-containing (120 mg/mL) (Sigma Aldrich, St. Louis, MO, USA) centrifuge tubes and diluted 1:2 with phosphate buffered saline (PBS) (Gibco, Life Technologies, Carlsbad, CA, USA). Information on sex, breed, and history was not available. Pigs were slaughtered at an age of approximately 6.5 months, chicken at an average age of 70 days, and cattle at an age of approximately >18 months. Exact age from individual animals is unknown, but all used study animals had reached at least late adolescence. Of this mixture, 35 mL was gently layered onto 15 mL Ficoll-Paque^TM Plus (GE Healthcare, Little Chalfont, UK) in 50 mL centrifuge tubes and centrifuged at 300× g without brake for 30 min. PBMCs, located in the middle of the obtained layers, were carefully aspirated and transferred into a fresh 50 mL centrifuge tube. Cells were washed three times with 40 mL PBS and cell numbers were determined via Tuerk solution (Sigma Aldrich, St. Louis, MO, USA) staining. Cells were used for assays immediately after isolation (no prior cryopreservation).