Sepmate

PBMC were isolated from Li-heparinized blood by centrifugation on a LymphoPrep™ gradient using SepMate™ centrifugation tubes (Stemcell™ Technologies Germany GmbH, Cologne, Germany). Cells were washed twice with PBS, and cell viability as well as cell concentration were determined using the trypan blue exclusion test. Cell suspensions of 1 × 10⁶cells/mL were prepared with RPMI, supplemented with 10% FCS, 1% Pen/Strep and glutamine. 1.5 mL of cell suspension was stimulated with 100 ng/mL LPS and incubated at 37°C in a humidified incubator with 5% CO₂/95% air atmosphere for 3, 24, or 48 h. Subsequently, the cells were centrifuged at 500× g for 5 min at 4°C. Cell free supernatants were collected, and stored at −80°C. Cell pellets were washed twice with ice cold PBS and stored at −80°C.

Peripheral blood mononuclear cells (PBMCs) were isolated using lymphoprep and SepMate (StemCell Technologies). Monocytes were isolated from PBMCs using EasySep™ Mouse Monocyte Isolation Kit. 500,000 monocytes/ml were cultured in complete RPMI with FBS supplemented with 50 ng/ml GM-CSF (Peprotech) plus 25 ng/ml IL-4 (StemCell Technologies) in 24-well plates for 3 days.

Whole blood (~350 mL) was collected from both male and female healthy adult donors via antecubital venipuncture into BD vacutainer, and Sodium Heparin blood collection tubes (BD, Franklin Lakes, NJ). Blood was diluted 2-fold in calcium- and magnesium-free phosphate-buffered saline (PBS pH: 7.2; Fisher Scientific, ​​Waltham, MA) within 30 min of collection. A single-step density gradient centrifugation was performed to isolate peripheral blood mononuclear cells (PBMCs) by slowly layering diluted blood (30 mL) over 15 mL of Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) contained within either a 50 mL SepMate™ (STEMCELL Technologies) or a 50 mL conical tube. Stepwise centrifugation at 1,200 x g for 10 min at room temperature with slow deceleration was performed. The buffy coat interface containing PBMCs was collected into 50 mL conical tubes and pelleted via centrifugation (1,400 RPM for 10 min). Residual red blood cells were removed with treatment with ACK Lysing Buffer (Gibco, Waltham, MA). PBMCs were then washed twice with 1X PBS, pelleted as above, and cryopreserved at -80°C in 90% (v/v) fetal bovine serum (FBS; R&D Systems, Minneapolis, MN) and 10% (v/v) dimethyl sulfoxide (Corning, Corning, NY) in cryovials labeled with each donor’s unique sample identification number, followed by transfer to liquid nitrogen storage within 24 h.

PBMCs were isolated using the density gradient medium, Lymphoprep™ from Stemcell Technologies (Vancouver, Canada) following their standard protocol. Briefly, 4 ml blood was diluted with 4 ml PBS and applied at the SepMate™ tubes (Stemcell Technologies) and spun at 1200xg for 20 min at RT in the centrifuge without brakes. The PBMC layer was collected and spun at 600xg for 8 min and washed two times in PBS by spinning at 600xg between each wash. After the last wash, the cells were counted and the cellular concentration was adjusted to 50 × 10⁶ cells/ml in PBS. Donor samples were treated with the LRRK2 inhibitor PFE-360 for one hour and the cells were hereafter lysed by adding 1/10 volume lysis buffer (5% Triton-X, 5% Digitonin, 10 mM EDTA) containing phosphatase and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The lysates from human PBMCs were subjected to SDS-PAGE and Western Blotting procedures as described above.

Healthy blood donations were obtained from the UW Carbone Cancer Center Translational Science Biocore Biobank under an approved IRB protocol. Blood was diluted 1:2 with PBS + 2% FBS and added to a 50 ml SepMate tube (Stemcell, Vancouver, BC) containing 15 ml of Lymphoprep (Stemcell, Vancouver, BC). The gradients were centrifuged at 1200g for 10 minutes and the PBMCs were poured into a new 50 ml conical tube. PBMCs were washed twice with PBS + 2% FBS before being stored frozen in RPMI + 40% FBS + 10% DMSO.

PBMC were isolated from Li-heparinized blood by centrifugation on a LymphoPrep^™ gradient using SepMate^™ centrifugation tubes (Stemcell Technologies, Cologne, Germany) and washed twice with PBS. Cell viability and cell concentration were determined using the trypan blue exclusion test.