TITLE: Dundee peripheral blood mononuclear cell (PBMC) isolation protocol (from whole blood)
AUTHORS: Adil R. Sarhan, Andrew J.M. Howden, Dario R. Alessi, Esther M. Sammler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">We describe a fast and efficient peripheral blood mononuclear cell (PBMC) isolation for LRRK2 kinase pathway analysis </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">Gain of kinase function mutations in the Leucine rich repeat kinase 2 (LRRK2) are associated with causing Parkinson’s disease. LRRK2 phosphorylates a subgroup of Rab GTPases and their phosphorylation levels mirror LRRK2 kinase activation status. Here, we describe a facile and robust method for isolating PBMCs by facilitated density gradient centrifugation, subsequent treatment with and without the specific LRRK2 kinase inhibitor MLi-2 to ensure that any effect seen is LRRK2 kinase dependent and cell lysis. PBMC lysates can then be used to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of its endogenous RabGTPase substrates in human peripheral blood neutrophils either by quantitative immunoblotting or targeted mass-spectrometry. The benefits of using PBMCs are that they are routinely used as a biomatrix for Parkinson’s research and their isolation procedure is significantly faster and easier than that for example of neutrophils or monocytes from peripheral blood. However, their heterogeneity in terms of cellular composition and highly variable expression levels of LRRK2 and its substrates including Rab10 make PBMC a suboptimal biomatrix for reliably measuring LRRK2 kinase pathway activity. A potential use could be target engagement studies in clinical trials targeting LRRK2. </div><style>
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[STEPS]
?. Collect 3 x  into BD Vacutainer sodium heparin 6ml tubes, green BD Hemogard Closure 13x100mm (BD, cat# 367876). Mix gently by inverting tubes 8 - 10 times.
[blood]
?. Add density gradient medium Ficoll-Paque PREMIUM to the SepMate™ tube (STEMCELL, Cat# 85450, 50 mL capacity) using a 20ml syringe with a large bore needle (at least 21G) by carefully adding the  through the central hole of the SepMate™ insert. Dispose of needle appropriately. The top of the density gradient medium will be above the insert. NOTE: Small bubbles may be present in the density gradient medium after pipetting. These bubbles will not affect performance.
15 mL
?. Using a 5 ml pipette, transfer  from the BD Sodium heparin tubes into a 50 ml Falcon tube and dilute at a 1:1 ratio by adding . Mix gently by inversion.
[blood]
[PBS with 2% FBS]
?. Keeping the SepMate™ tube vertical, add the diluted blood sample by pipetting it down the side of the tube. Take care not to pour the diluted blood sample directly through the central hole that lies in the centre of the insert disk.
?. Centrifuge at , with the brake ON.
Centrifuge: 1200 34, 10 min
?. After centrifugation, carefully pipette off the top plasma transparent layer and discard appropriately.
?. Using a new pipette, collect the remainder of the sample above the insert disk (which contains the PBMCs) into a 15 mL falcon tube.
?. Top up the tube to . Mix gently by inversion.
[PBS with 2% FBS]
?. Centrifuge at , brake ON, to pellet the PBMCs.
Centrifuge: 1000 34, 2 min
?. Carefully discard the supernatant without disturbing the PBMC pellet.
?. Carefully resuspend the cell pellet in .
[PBS with 2% FBS]
?. Centrifuge at , brake ON to pellet the PBMCs.
Centrifuge: 1000 34, 2 min
?. During the centrifugation Step 12, take MLi-2 inhibitor stock solution (200 µM / 1000x) out of the  freezer, and leave at  to thaw for subsequent use (Steps 19 and 21)
-80 °C
-80 Room temperature
?. Immediately after the centrifugation, carefully discard the supernatant without disturbing the PBMC pellet.
?. Resuspend the PBMC cell pellet in  by gently pipetting cells up and down 4 times.
[PBS with 2% FBS]
?. Divide resuspended PBMCs equally into 2 tubes ( in each).
5 mL
?. Label one tube “DMSO” and other tube “MLi-2”.
?. To “DMSO” labelled tube, add  and mix gently by pipetting up and down 2 times with a 10 ml pipette.
[DMSO]
?. To “MLi-2” labelled tube, add  and mix gently by pipetting up and down 2 times with a 10 ml pipette.
[of 200 µM MLi-2 stock solution (final concentration 200 nM)]
?. During the incubation period:
?. Incubate samples for  at . Mix gently by inversion every  during the incubation period.
-80 Room temperature
?. Remove 0.5 M DIFP stock from  freezer and place in fume hood .
-80 °C
on ice
?. Remove 1 mg/ml Microcystin-LR stock from freezer and place at  to thaw.
-80 °C
-80 Room temperature
?. Defrost an aliquot () of the lysis buffer by taking it out of the freezer allow to defrost at and then place it  for subsequent use (Step 27)
0.5 mL
-80 Room temperature
on ice
?. Prepare  containing  and call this “DMSO Resuspension Buffer”
[PBS with 2% FBS]
[DMSO]
?. Prepare  containing  and call this “MLi-2 Resuspension Buffer”
[ PBS with 2% FBS]
[200 µM MLi-2 stock solution]
?. After the  incubation, centrifuge both tubes at . The setting for acceleration and deceleration is both 5 using a Beckman Coulter Allegra X-15R Centrifuge (maximum is 10) to pellet the PBMCs.
Centrifuge: 335 34, 5 min
?. Carefully discard the supernatant in each tube without disturbing the PBMC pellets.
?. For the DMSO labelled sample gently resuspend pellet in  and transfer to Eppendorf tube labelled “DMSO”.
[“DMSO resuspension buffer”]
?. For the MLi-2 labelled sample gently resuspend pellet in  and transfer to Eppendorf tube labelled “MLi-2”.
[“MLi-2 resuspension buffer”]
?. Centrifuge both tubes at .
Centrifuge: 300 34, 3 min
?. During this time proceed with preparing the lysis buffer: In the fume hood carefully to the  add  as well as . Mix and leave  until use. DIFP should be added to lysis buffer within  of cell lysis (Steps 31 and 32) as DIFP relatively unstable in aqueous solution.
[lysis buffer]
[0.5 M DIFP solution]
[1 mg/ml microcystin-LR]
on ice
?. Return 0.5M DIFP to  freezer.
-80 °C
?. Immediately after the centrifugation in Step 26, carefully discard supernatant by using a pipette while taking care not to disturb the PBMC pellets.
?. Place the tubes with the PBMC cell pellets .
on ice
?. Immediately add  containing DIFP to the “DMSO” labelled tube. Using a 100-200 µL pipette, re-suspend the cell pellet by pipetting up and down until all cells are lysed (5-10 times).
[lysis buffer]
?. Next add  to the “DMSO” labelled tube. Using a 100-200 µL pipette, re-suspend the cell pellet by pipetting up and down until all cells are lysed (5-10 times).
[lysis buffer containing DIFP]
?. Incubate cell lysates  for . Place “DMSO” and “MLi-2” labelled tubes into a centrifuge and remove cell debris by centrifugation at .
on ice
Centrifuge: 14000 33, 15 min, 4 10
?. Transfer ”DMSO” and “MLi-2” supernatants (containing PBMC lysates) into new Eppendorf tubes. Discard debris pellet.
?. Immediately snap freeze samples in liquid nitrogen.
?. Store samples at  until further use or shipment on dry ice keeping samples frozen at all times.
-80 °C