Ficoll hypaque

Peripheral blood samples (73 ALS and 48 controls) were collected in lithium-heparin tubes (Sarstedt). PBMCs were prepared by Ficoll–Hypaque (Biochrom) density centrifugation, then washed with phosphate-buffered saline (PBS) and counted manually. Serum samples for cytokine analysis were collected in 7.5 ml tubes (Sarstedt) and centrifuged at 2000g for 10 min, aliquoted and frozen at −80 °C within 20 min after collection.
CSF samples (16 ALS and 10 controls) were centrifuged at 250 g for 10 min at 4 °C within 20 min after collection, the cell pellet was collected and immediately processed by flow cytometry.

Absolute blood cell numbers in patient samples were determined on a Sysmex XN 1000/9000 clinical diagnostics system according to the manufacturer's recommendations. PBMCs were isolated from heparinised human whole blood by Ficoll‐Hypaque (Biochrom, Berlin, Germany) gradient in SepMate tubes (STEMCELL, Vancouver, Canada) within 6 h from blood draw. Cells were further analysed directly after isolation. Sera were stored at −80°C until further use.

Peripheral blood mononuclear cells (PBMC) were isolated from the leukocyte concentrates or from EDTA blood of ovarian patients by Ficoll-Hypaque™ PLUS (Cytiva, Uppsala, Sweden) density gradient centrifugation. Cells were washed in PBS, and resuspended in RPMI 1640 (Gibco, Paisley, Scotland) supplemented with 2 mM L-glutamine, 25 mM Hepes, 100 U/mL penicillin, 100 μg/mL streptomycin (PanReac AppliChem, Darmstadt, Germany), 10% FCS (Thermo Fisher Scientific, Langenselbold, Germany) (complete medium). Tumor-infiltrating cells (TIL) were isolated from the dissociated tumor tissue described under 2.2. Digested cell suspension was passed after the gentle MACS through a 100 µm cell strainer (Falcon, BD Biosciences, Heidelberg, Germany), and centrifuged at 481 ×g for 5 min. TIL and tumor cells were separated by Ficoll-Hypaque (Biochrom, Berlin, Germany) density gradient centrifugation followed by an adherence step for several hours.
The percentage of Vδ1 T cells within PBMC ranged between 0.1 and 3% (median 0.5%), and in TIL between 0.5 and 5% (median 1%), whereas the percentage of Vδ2 T cells within PBMC ranged between 0.1 and 10% (median 1.7%), and in TIL between 0.1 and 2.5% (median 0.9%).

PBMCs were isolated from peripheral blood from patient 1 and a healthy control by density gradient centrifugation using Ficoll-Hypaque (Biochrom AG, Berlin, Germany) and immediately subjected to multi-colour flow cytometric analysis. For B cell phenotyping, 10⁶ PBMCs were stained with fluorescent-dye-labelled monoclonal antibodies (mAbs) specific for CD20, CD27 and IgD. Stained cells were first gated for CD20⁺ (total B cells), followed by CD27/IgD dot-plot analysis identifying CD20⁺CD27⁻IgD⁺ naïve, CD20⁺CD27⁺IgD⁺ non-switched memory B cells, and CD20⁺CD27⁺IgD⁻ isotype class-switched memory B cells [16 (link)]. For detection of T lymphocytes, 10⁶ PBMCs were stained with fluorescent-dye-labelled mAbs specific for CD3, CD4, CD8 and CD45RO. Stained cells were first gated for CD3 (total T cells), followed by CD4/CD45RO and CD8/CD45RO dot-plot analysis to identify CD3⁺CD4⁺ T-helper cells, CD3⁺CD4⁺CD45⁺ memory T-helper cells, CD3⁺CD4⁺CD45RO⁻ naïve T-helper cells, CD3⁺CD8⁺ cytotoxic T cells, CD3⁺CD8⁺CD45⁺ memory cytotoxic T cells and CD3⁺CD8⁺CD45RO⁻ naïve cytotoxic T cells. All mAbs were purchased from BD Pharmingen (Heidelberg, Germany). Fluorescence-activated cell sorting (FACS) data acquisition was performed with a FACS Calibur™ cytometer and analysed with CellQuest™ software (BD Biosciences, Heidelberg, Germany).

CLL and ALL samples were collected with informed consent and in accordance with the Helsinki declaration on approval from the Ethics Committee of the University of Erlangen-Nuremberg (Table S2).
PBMCs were isolated from heparinized blood samples by centrifugation over a Ficoll-Hypaque layer (Biochrom, Berlin, Germany) of 1.077 g/ml density. For CLL samples, T cells and monocytes were removed with anti-CD2 and anti-CD14 magnetic beads (Dynabeads M450, Dynal, Oslo, Norway).

Blood from healthy donors was acquired with informed consent according to the Helsinki Declaration and the local ethical board. PBMCs were isolated by density-gradient centrifugation with Ficoll/Hypaque (Biochrom) and frozen in FCS supplemented with 10% DMSO. For genetic modification, the cells were thawed and CD8 T cells were isolated via negative selection with the Dynabeads Untouched Human CD8 T Cells kit (Thermo Fisher Scientific).