The overall goal of this procedure is to provide a method to assess endogenous LRRK2 kinase activity in human peripheral blood. This is achieved by monitoring LRRK2-mediated phosphorylation of Rab proteins, employing Michael J.Fox Foundation phospho-specific Rab monoclonal antibodies. Here, we focus on human peripheral blood neutrophils as a constitute of homogenous site population with high expression levels of both Rab2 and Rab10.
The neutrophil isolation is based on a negative selection that within 40 minutes of finished action allows isolation of 99%pure and viable neutrophils while also yielding high amount of protein levels. Collect 10 milliliter of blood into a blood collection tube. Mix gently by inverting tubes seven to eight times.
Transfer 10 milliliters of blood into a 50 milliliter conical tube. Add to blood 100 microliters of EDTA stock solution 1, 0.1 molar EDTA PBS Solution, mix gently. Add 500 microliters isolation cocktail, 50 microliters per milliliter from the neutrophil isolation kit to the whole blood sample.
Vortex the magnetic beads from the neutrophil isolation kit for 30 seconds before use in order to resuspend very fine magnetic beads. Add 500 microliters magnetic beads to the blood sample and mix gently by inverting the tube several times. Incubate at room temperature for five minutes.
Top up tube to 50 milliliter with EDTA stock solution 2. This is one millimolar EDTA PBS Solution. Mix by very gently pipetting up and down two to three times.
Place the tube into the magnet and remove the lid to avoid subsequent agitation of the tube. Incubate for 10 minutes at room temperature. Carefully pipette enriched cell suspension that contains neutrophils into a new 50 milliliter conical tube.
Do not 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 10 milliliters of the red blood cell suspension behind at the bottom of the tube. Vortex magnetic beads for 30 seconds before use and add 0.5 milliliter magnetic beads to the tube containing the enriched neutrophils.
Mix gently by inverting the tube. Incubate at room temperature for five minutes. Place the tube into the magnet and remove the lid to avoid subsequent agitation.
Incubate for five minutes at room temperature. Carefully pipette the enriched cell suspension that contains neutrophils in a new 50 milliliter conical tube. Do not touch the side of the tube that is in contact with the magnet.
Leave approximately five milliliter of the suspension at the bottom of the tube. To ensure complete removal of magnetic beads from cell mixture, place tube containing enriched cells into the magnet. Incubate for 10 minutes at room temperature.
Carefully pipette the enriched cell suspension that now contains pure neutrophils into a new 50 milliliter conical tube. Do not touch the side of the tube that is in contact with the magnet. Leave approximately five milliliter of suspension at the bottom of the tube.
Top up isolated cells with one millimolar EDTA stock solution 2 to a final volume of approximately 41 milliliters. Pipette up and down to mix. Divide solution equally into two tubes with approximately 20 milliliters in each tube.
Centrifuge both tubes at 335G for five minutes. During the centrifugation step, take MLi-2 inhibitor stock, 200 micromolar slash 1000X concentration out of the minus 80 degree freezer and leave at room temperature for subsequent use. Immediately after the centrifugation step and without agitation of the tubes, pour off the supernatant without disturbing the neutrophil pellets.
resuspend each cell pellet in 10 milliliters of cell culture medium at room temperature by gently pipetting cells up and down four times. Label one tube DMSO and the other tube MLi-2. To DMSO labeled tube add 10 microliter of DMSO and add 10 microliters of 200 micromolar MLi-2 stock solution by gently pipetting up and down to mix.
Incubate samples for 30 minutes at room temperature. Mix gently by inversion every 10 minutes during the incubation period. During the 30 minute LRRK2 kinase inhibitor treatment remove 0.5 molar DIFP stock solution from the minus 80 degree freezer and place in fume hood on ice.
Then, move one milligram per milliliter Microcystin-LR stock solution from the minus 80 degree freezer and place at room temperature to thaw. Defrost an aliquot, 0.25 milliliter of the lysis buffer by taking it out of the freezer, allow to defrost at room temperature and then place it on ice for subsequent use. Prepare one milliliter of RPMI medium containing one microliter of DMSO call this DMSO Resuspension Buffer.
Prepare one milliliter of RPMI medium containing one microliter of 200 micromolar MLi-2 or alternative LRRK2 kinase inhibitor and call this MLi-2 Resuspension Buffer. After the 30 minute incubation period, centrifuge both tubes at 335G for five minutes, as before. Carefully discard the supernatant in each tube without disturbing the neutrophil pellet.
For the DMSO labeled tube, gently resuspend pellet in one milliliter of DMSO Resuspension Buffer. And for the MLi-2 labeled tube, resuspend pellet in one milliliter of MLi-2 Resuspension Buffer. Transfer resuspended cell pellets to corresponding centrifugation tubes labeled DMSO and MLi-2 and centrifuge both tubes at 335G for three minutes.
During the centrifugation step, prepare the lysis buffer. In the fume hood, carefully add 0.25 microliters of 0.5 molar DIFP solution, as well as 0.25 microliters of one milligram per milliliter microcystin-LR to the 0.25 milliliter lysis buffer. Mix and leave on ice until use.
Immediately after the centrifugation, carefully and completely remove all supernatant with a pipette without disturbing the neutrophil pellet and place tubes on ice. Immediately add 100 microliters of lysis buffer containing DIFP and microcystin-LR to each tube. Using a 100 to 200 microliter pipette, resuspend the cell pellets by pipetting up and down about five to 10 times.
Lie cells on ice for 10 minutes. Then, centrifuge tubes at 20, 000 times G for 15 minutes at four degrees celsius to remove cell debris. Transfer DMSO and MLi-2 supernatant containing neutrophil lysates into new centrifugation tubes.
Discard debris pellets. Neutrophil lysates are now ready for use or can be snap frozen in liquid nitrogen and stored at minus 80 degrees for future analysis. Using data from the publicly available import database, this graph shows that an abundance of the LRRK2 and Rab10 proteins is particularly high in human peripheral blood neutrophils and monocytes.
Isolating human peripheral blood neutrophils with this procedure results in a high impure yield of cells. The table at the top shows the total number of cells isolated from 10 milliliter of peripheral blood from three healthy donors. Also shown are purity and viability of the isolated cells, as well as total protein lysate yield.
After treatment with and without the LRRK2 kinase inhibitor, MLi-2, neutrophils are lysed in the presence of the potent protease inhibitor, DIFP. For figure B on the left hand side, 10 micrograms of whole cell extracts per lane were subjected to multiplex quantitative immunoblot analysis with the indicated antibodies. The figure on the right, C, demonstrates the importance of DIFP for prevention of proteolytic degradation, in particular of the large LRRK2 protein.
In comparison, high concentrations of PMSF alone are less effective. Here is showed that LRRK2-controlled Rab10 phosphorylation is significantly increased by about threefold in peripheral blood neutrophils derived from patients with hereditary Parkinson's disease harboring a heterozygous VPS35 D620N mutation when compared to controls. The top graph shows the multiplex immunoblot analysis and the bottom, the quantification thereof.
This suggests that the VPS35 D620N mutation activates LRRK2 kinase path of activity by a yet unknown mechanism. In summary, we present a facile and robust assay to measure LRRK2 kinase pathway activity by monitoring Rab protein phosphorylation, such as Rab10, in human peripheral-derived neutrophils, this method allows us to assess LRRK2 pathway activity and determine how mutations, disease states or inhibitors modulate LRRK2 pathway activity.
