TITLE: Dundee neutrophil isolation protocol (from whole blood)
AUTHORS: Ying Fan, 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 immunomagnetic negative isolation method to purify peripheral blood neutrophils for LRRK2 kinase pathway analysis in humans</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>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 peripheral blood neutrophils by immunomagnetic negative selection, 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. Neutrophil lysates can then be used to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of its endogenous Rab GTPase substrates either by quantitative immunoblotting or targeted mass-spectrometry. The benefit of using peripheral blood neutrophils is that they are an abundant and homogenous white blood cell population that express relatively high levels of LRRK2, Rab10 and other Rab GTPases. The downside is their high intrinsic elastase serine protease activity that necessitates using the neurotoxic, but efficient serine protease inhibitor Diisopropyl fluorophosphate (DIFP) when lysing the cells. Using this assay, we have demonstrated significantly elevated LRRK2 kinase pathway activity in human peripheral blood neutrophils of patients carrying specific mutations such as LRRK2 R1441G (doi:https://doi.org/10.1101/2021.01.28.21249614) and VPS35 D620N (PMID:</span><a href="https://www.ncbi.nlm.nih.gov/pubmed/29743203" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">29743203</span></a><span>). This assay can also be used for pharmacodynamic and target engagement studies of small molecule LRRK2 kinase inhibitors.  </span></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
?. Collect  into BD Vacutainer. Ensure  is collected by not prematurely discontinuing blood collection. Mix gently by inverting tubes 7-8 times.
[blood]
[blood]
?. Transfer  into a 50 mL falcon tube.
[blood]
?. Add to blood . Mix gently.
[“EDTA Stock Solution 1” (0.1M EDTA-PBS solution)]
?. Add  from the neutrophil isolation kit to the whole blood sample.
[“isolation cocktail solution”]
?. Vortex the “RapidSpheres magnetic beads” from the neutrophil isolation kit for  before use in order to resuspend very fine magnetic beads.
?. Add  to the blood sample.
[RapidSpheres magnetic beads]
?. Mix blood sample gently by inverting the tube and incubate at  for .
0 Room temperature
?. Top tube up to 50 ml with “EDTA Stock Solution 2” (1 mM EDTA-PBS solution).
?. Mix by very gently pipetting up and down 2-3 times.
?. Place the tube into the magnet and remove the lid to avoid subsequent agitation of tube. Incubate for  at .
0 Room temperature
?. Carefully pipette the enriched cell suspension that contains neutrophils into a new 50 ml falcon tube. Take care not to touch the side of the tube that is in contact with the magnet and avoid collection and perturbation of the red blood cells at the bottom of the tube (leave approximately  behind at the bottom of the tube).
[red blood cell suspension]
?. Add  to the tube from Step 11.
[RapidSpheres magnetic beads]
?. Mix blood sample gently by inverting the tube and incubate at  for .
0 Room temperature
?. Place the tube into the magnet, remove the lid, and leave for  at .
0 Room temperature
?. Carefully pipette the enriched cell suspension that contains neutrophils into a new 50 ml falcon tube. Take care not to touch the side of the tube that is in contact with the magnet. Leave approximately  at the bottom of the tube.
[suspension]
?. To ensure complete removal of magnetic beads from cell mixture, place the new tube from Step 15 into the magnet, remove the lid, and leave for  at .
0 Room temperature
?. Carefully pipette the enriched cell suspension that now contains pure neutrophils into a new 50 ml falcon tube. Take care not to touch the side of the tube that is in contact with the magnet. Leave approximately  at the bottom of the tube.
[suspension]
?. The neutrophils will be in a volume of 30-35 ml at this stage.
?. Top up isolated cells with 1 mM EDTA stock solution 2 to a final volume of approximately . Pipette up and down to mix. Divide solution equally into 2 tubes with approximately  in each tube.
41 mL
20 mL
?. Centrifuge both tubes at . The setting for acceleration and deceleration is both 5 using a Beckman Coulter Allegra X-15R Centrifuge (maximum is 10).
Centrifuge: 335 34, 5 min
?. During this centrifugation take	MLi-2 inhibitor stock (200 µM / 1000X concentration) out of the  freezer, leave	 at  for subsequent use (Steps 26 and 28).
-80 °C
0 Room temperature
?. Immediately after the centrifugation, carefully discard the supernatant without disturbing the neutrophil pellet.
?. Resuspend each cell pellet in  at by gently pipetting cells up and down 4 times.
[RPMI medium]
0 Room temperature
?. 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.
[200 µM MLi-2 stock solution (final concentration 200 nM)]
?. Incubate samples for  at . Mix gently by inversion every  during the incubation.
0 Room temperature
?. During the incubation period,
?. 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 leave at 	to thaw.
-80 °C
-80 Room temperature
?. Defrost an aliquot () of the lysis buffer by taking it out of the freezer, allowing it to defrost at  and then placing it  for subsequent use (Step 34).
0.25 mL
-80 Room temperature
on ice
?. Prepare  containing  and call this “DMSO Resuspension Buffer”.
[RPMI medium]
[DMSO]
?. Prepare  containing  and call this “MLi-2 Resuspension Buffer”.
[RPMI medium]
[200 µM MLi-2]
?. After the 30 min 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 neutrophils.
Centrifuge: 335 34, 5 min
?. Carefully discard the supernatant in each tube without disturbing the neutrophil pellet.
?. 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 (Step 38 and 39) 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 stock to  freezer at this stage.
-80 °C
?. After the centrifugation in Step 33, immediately proceed to carefully remove all supernatant with a pipette without disturbing the neutrophil pellets.
?. Place the tubes with the neutrophil cell pellets
on ice
?. Immediately 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]
?. Next add  to the “MLi-2” labelled tube. Using a 100-200 µL pipette and 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 neutrophil 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