Common use of Materials and Methods Clause in Contracts

Materials and Methods. Materials T-cell leukemia cells (Jurkat, Clone E6-1, ATCC® TIB152™) were provided by Leiden University Medical Centre (LUMC) as frozen 1-ml aliquots at a total cell concentration of 107 cells/ml, and were stored at -140 oC in the freezer prior to usage. The Jurkat cells were formulated in high-glucose RPMI 1640 (RPMI medium; ThermoFisher, Waltham, USA) supplemented with 10% fetal bovine serum (Life Technologies, USA) and 10% dimethyl sulfoxide (DMSO) (Life Technologies, USA). Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation and low-protein binding collection tubes were purchased from ThermoFisher (Waltham, USA). Sterile 5-ml Eppendorf tubes were purchased from VWR (Ismaning, Germany). Sample preparation Jurkat cells used in this study as model T- cells were thawed and freshly prepared in RPMI medium prior to analysis. Frozen cell aliquots were thawed at 36oC and resuspended in ca. 40 ml of RPMI medium. To remove residual fetal bovine serum and DMSO, the cell suspension was centrifuged at 300 rcf for 10 minutes at 20oC. The supernatant was removed and the pellet was resuspended in 10 ml of RPMI medium, unless otherwise stated. The mean concentration of (live and dead) cells was 477,188 ± 85,914 per ml with a mean viability of 81% ± 9% (n=8) as determined by hemocytometry (described below), unless otherwise stated. Cell containing samples were measured up to 4 hours post thawing, a time window during which the cell viability was not affected (data not shown). Dynabeads were diluted to an intermediate stock concentration of 106 beads/ml (based on the dilution factor of the nominal Dynabead concentration) in RPMI medium and stored at 2-8 oC for up to 1 month. The required volume of the intermediate stock was added to cell samples to reach the desired Dynabead concentrations. Reference concentration of Dynabeads stated in the results section is the expected concentration of Dynabeads in the sample derived from dilution calculations and the original bead concentration stated by the manufacturer. It must be noted that the manufacturer does Particulates impurities in CBMPs traced by FIM-CNN not use FlowCam for quantification of Dynabeads; therefore, a systematic deviation between reference concentrations and measured concentrations should be anticipated. Hemocytometry Cell viability and total cell concentration were determined by using a Bright-Line hemocytometer glass (Merck, Darmstadt, Germany) and an Axiostar Plus microscope (Zeiss, Jena, Germany) with 10x magnification (Zeiss, Jena, Germany). The washed cell suspension was diluted 2-fold with a sterile-filtered 0.4% trypan blue solution (Merck, Darmstadt, Germany). Next, 10 µl of the mixture was placed in the hemocytometer and at least 100 cells were counted (both viable – not stained, and non-viable – stained cells), following the manufacturer´s recommendations. Flow imaging microscopy (FIM) For characterization of micron-sized particles, a FlowCam 8100 (Fluid Imaging Technologies, Scarborough, USA) equipped with an 80-µm flow cell and a 10x objective was used. The instrument was operated by using a VisualSpreadsheet software (v4.10.8). Analysis was performed by using a flowrate of 0.18 ml/min and the detection thresholds were set to 17 for dark pixels and 15 for light pixels. Images were taken with a high- resolution CMOS camera (1920x1200 pixels) at 27 frames per second. In total, a sample volume of 0.5 ml was analyzed with an efficiency of approximately 70% (i.e., the measured sample volume was ca. 0.35 ml). Cleaning steps between sample measurements involved thorough flushing of the flow cell with 2% Hellmanex III and highly purified water. Diameters are reported as equivalent spherical diameter (ESD) and filters were not applied for imaging pre-processing. Samples were measured in triplicate or sextuplicate, unless otherwise stated.