May 09, 2024

Public workspaceIsolation of lysosomes using the Tagless LysoIP method in PBMCs V.1

DOI
dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1
Isolation of lysosomes using the Tagless LysoIP method in PBMCs
  • 1Aligning Science Across Parkinson's;
  • 2Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Dario R Alessi
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1
Protocol Citation: Daniel Saarela, Esther Sammler, Dario R Alessi, Francesca Tonelli 2024. Isolation of lysosomes using the Tagless LysoIP method in PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 07, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59146
Keywords: Purification of lysosomes, Isolation and homogenisation of PBMCs, Tag-less lysosomes, TMEM192 immunoprecipitation, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
Abstract
Molecular homeostasis in cells is regulated in part by protein degradation, which is facilitated by the proteasome and lysosomal proteolysis. Lysosomes are membrane bound organelles involved in the turnover of proteins, metabolites and lipids. Recent literature implicates lysosomal dysfunction to be a feature of many a disease, including neurodegenerative diseases. Focused investigation of lysosomal content (proteome/lipidome/metabolome) in disease states could lead to the discovery of novel therapeutics and disease mechanisms. Here we describe our method to isolate peripheral blood mononuclear cells (PBMCs) and perform rapid isolation of intact, tag-less lysosomes from PBMC homogenates using ball-bearing cell breakers and anti-TMEM192 -antibody coupled to magnetic beads. First, cells are broken with physical sheering force as the cell suspension passes through a narrow gap within the cell-breaker, leading to plasma membrane rupture but due to their small size, lysosomes remain intact (Figure 1). Then, the cell homogenate is incubated with the antibody-coupled magnetic beads to allow for rapid immunopurification of lysosomes by binding to the transmembrane protein of lysosomes TMEM192 (Figure 3). The purified lysosomes can be processed and analysed with a variety of techniques including immunoblotting analysis, proteomic/lipidomic/metabolomic tools, fluorescence-activated cell sorting and GCase activity assay. The immunopurification protocol is very fast, less than 15 minutes from start of the incubation with the beads to washed, pure lysosomes. The same protocol can also be used to immunopurify lysosomes from commonly cultured cells such as mouse embryonic fibroblast, HEK293 and A549 cells.

Image Attribution
Figure 1 and Figure 3 were created using BioRender.com
Guidelines
Supporting figures

Figure 1

Figure 1. Isolation and homogenisation of PBMCs.
Figure 1. Isolation and homogenisation of PBMCs.
Graphical overview of the workflow of PBMC isolation and homogenisation. Note that images are not to scale, and the homogenising is performed with a different instrument, as outlined in Figure 2.


Figure 2.
Figure 2. Isobiotec cell-breaker.
Figure 2. Isobiotec cell-breaker.
(A) Isobiotec cell-breaker with one side panel open and a 10 μm-clearance metal ball with its container.
(B) Example of assembled cell-breaker being used to homogenise cells TemperatureOn ice .
Figure 3.


Figure 3. TMEM192 immunoprecipitation from PBMC homogenates.
Figure 3. TMEM192 immunoprecipitation from PBMC homogenates.
(A) Graphic describing the design of anti-TMEM192 antibody-bound beads attached to a lysosome.
(B) Beads are incubated with cell homogenate containing lysosomes for Duration00:05:00 . After immunoprecipitation, the beads are immobilised with magnets and washed with KPBS. Lysosomes attached to the beads are lysed and the supernatant containing lysosomal contents is collected for analysis.

Figure 4.
Figure 4. Immunoblot analysis of Tagless LysoIP in PBMCs
Figure 4. Immunoblot analysis of Tagless LysoIP in PBMCs
Immunoblot analysis confirming enrichment of lysosomes with Tagless Lyso-IP in PBMCs. PBMCs were treated with 0.5 mM diisopropylfluorophosphate (DIFP) to prevent rapid degradation of proteins. Whole-cell lysates (2 µg) as well as the resuspended immunoprecipitates (IPs) (2 µg) were subjected to immunoblotting with the lysosomal (LAMP1, TMEM55B, LAMTOR1), Golgi (GM130), cytosolic (α-tubulin, GAPDH) and mitochondrial (HSP60) markers. The data shown is from 3 healthy male donors.




Materials
Reagents

  1. ReagentGibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190169
  2. Foetal Bovine Serum
  3. ReagentDynabeads™ MyOne™ Epoxy, for OEM and industrial use onlyThermo FisherCatalog #34001D coupled to anti-TMEM192 antibody.
  4. ReagentFicoll-Paque PREMIUM density gradient mediaCytivaCatalog #17544652
  5. Diisopropyl fluorophosphate (DIFP) ReagentDiisopropylfluorophosphateMillipore SigmaCatalog #D0879
  6. KPBS Buffer (homogenisation buffer):
AB
 KCL136 mM
KH2PO410 mM
Adjust to Ph7.25 with KOH.
Note
Note: On the day of use, add Roche cOmplete protease inhibitor cocktail tablet (REF# 11873580001) and Roche PhosSTOP tablet (REF# 04906837001).
ReagentRoche cOmplete™ EDTA-free Protease Inhibitor CocktailMillipore SigmaCatalog #11873580001
ReagentRoche PhosSTOP™Millipore SigmaCatalog #4906837001

Equipment

Equipment
BELLY DANCER ORBITAL SHAKER
NAME
Shaker
TYPE
ORBITAL
BRAND
BDRAA115S
SKU
https://www.ibisci.com/products/belly-dancer-shaker
LINK

Equipment
DynaMag™-2 Magnet
NAME
Magnet
TYPE
DynaMag™
BRAND
12321D
SKU
https://www.thermofisher.com/order/catalog/product/12321D
LINK

  1. Isobiotec Cell-Breaker, isobiotec Vertriebs UG
  2. Stripetter/stripette gun and stripettes
  3. Set of Gilson pipettes P10, P200, P1000
  4. Microcentrifuge with thermostat
Equipment
Microcentrifuges, ventilated/refrigerated, Micro Star 17 / 17R
NAME
Microcentrifuge
TYPE
VWR®
BRAND
521-1647
SKU
https://in.vwr.com/store/product/8306728/microcentrifuges-ventilated-refrigerated-micro-star-17-17r
LINK


Consumables
  1. ReagentSafeSeal reaction tube 1.5 ml PP PCR Performance Tested Low protein-bindingSarstedtCatalog #72.706.600 .
  2. ReagentConical tube, 15 mlgreiner bio-oneCatalog #188271 .
  3. Reagent50 ml centrifuge tubes greiner bio-oneCatalog #227261 .
  4. Standard 1ml and 200µl Pipette tips (Greiner bio-one. Catalog# 686271 and ReagentPIPETTE TIP 10 - 100 µL SUITABLE FOR EPPENDORF 96 PIECES / ST RACKgreiner bio-oneCatalog #685261 , respectively).
  5. ReagentSyringe Filter Minisart High Flow PES 28 mm 0.45µm non-sterile 500 pc/PAKSartoriusCatalog #ST16537-Q )
  6. 1ml Terumo Syringe without needle (ReagentTerumo® Syringe 3-part SyringeTerumoCatalog #MDSS01SE )
  7. ReagentBecton Dickinson Disposable needles 21G x 1 1/2 inch Becton-DickinsonCatalog #304432
  8. Syringe without needle (10ml) (Medicina. REF# IVS10. LOT# 19111004)
  9. ReagentSyringe PP/PE without needleSigma AldrichCatalog #Z116866
  10. ReagentSepMate™-50 (IVD) 100 Tubes Stemcell TechnologiesCatalog #85450
  11. ReagentBD Vacutainer 10ml Glass Sodium Heparin Tubes (100)Becton-DickinsonCatalog #368480
















Methods
Methods
26m 30s

Note
According to local UK regulation, we undertake all manipulations and pipetting of human blood in category 2 biological safety cabinet.
Prepare a Concentration0.5 Molarity (M) stock solution of DIFP by diluting in isopropanol under a fume hood. Stock solution can be stored in Temperature-80 °C until needed.
Note
Note: DIFP is extremely toxic and should be handled with extra care, under a fume hood and wearing double layers of gloves. All waste from this step must be disposed of into 2% (w/v) NaOH solution in distilled water, which inactivates DIFP.

Critical
Toxic
Collect blood into a BD Vacutainer sodium heparin 10-mL tubes.
Note
Note: Amount17 mL of blood is the maximum amount that fits a single SepMate tube.


Add density gradient medium to the SepMate tube using a 20-mL syringe with a large bore needle (at least 21G) by carefully dispensing Amount15 mL through the central hole of the SepMate insert.
Note
Note: The density gradient should settle slightly above the insert.

Pipetting
Transfer the blood from the BD Sodium heparin tubes into a 50-mL Falcon tubes.
Dilute the blood by adding an equivalent volume of PBS containing 2% (v/v) FBS to achieve a 1:1 ratio. Mix gently by inversion.
Mix
Gently pipette the blood into the SepMate tube containing the density gradient medium.
Pipetting
Centrifuge for Centrifigation1200 x g, 4°C, 00:10:00 .

Centrifigation
Decant the top layer (containing PBMCs) to a new 50-ml Falcon tube, leaving the red bottom layer in the tube.
Note
Note: Decant quickly to avoid contamination of your sample with red blood cells.

Top up the Falcon tube from Step 8 with PBS up to Amount50 mL and centrifuge the cells at Centrifigation450 x g, 4°C, 00:05:00 . Discard the supernatant.
Centrifigation
Repeat Step 9: Top up the Falcon tube from Step 8 with PBS up to Amount50 mL and centrifuge the cells at Centrifigation450 x g, 4°C, 00:05:00 . Discard the supernatant.
Note
Note: This step is performed to wash out the remaining plasma.

Centrifigation
After last wash with PBS, resuspend the pellet in Amount1 mL KPBS and transfer to a low-binding Eppendorf tube.


Note
Steps from here on are performed on standard bench. This work should be done TemperatureOn ice .

Aliquot Amount100 µL of pre-prepared TMEM192-beads per IP into a fresh Eppendorf tube.
Note
Note: A protocol for preparing anti-TMEM192 antibody-conjugated beads is available on protocols.io.

Immobilize the beads by placing the tubes from Step 12 into a Dyna-Mag tube holder for Duration00:00:30 .

Remove the supernatant using a pipette and gently resuspend the beads in Amount1 mL of KPBS.

Pipetting
Repeat Step 13: Immobilize the beads by placing the tubes from Step 12 into a Dyna-Mag tube holder for Duration00:00:30 .
Remove the supernatant using a pipette.
Pipetting
Gently resuspend the beads in n x Amount100 µL of KPBS, where n = number of samples to be subjected to immunoprecipitation.

Aliquot the beads from Step 17 into fresh low-binding Eppendorf tubes (Amount100 µL of slurry each). Leave the tubes TemperatureOn ice until use (Step 27).

Centrifuge the cells from Step 11 at Centrifigation1500 x g, 4°C, 00:02:00 to compare total cell pellets between samples. Note down the size and colouring of the pellets.
Note
Note: The colour of cell pellets is an indication of red blood cell contaminants; the size of cell pellets is an indication of the expected total protein yield.


Centrifigation
Resuspend the cells in Amount800 µL KPBS and add DIFP to a final concentration of Concentration0.5 millimolar (mM) (1:1000 dilution of stock solution). Leave the tubes TemperatureOn ice .
Note
Note: DIFP is extremely toxic. This step must be performed under a fume hood and with double layers of gloves. All waste from this step must be disposed of into 2% (w/v) NaOH solution in distilled water, which inactivates DIFP.



Toxic
Transfer Amount50 µL of whole cell sample (WC) from Step 20 to a fresh Eppendorf tube and place TemperatureOn ice .

To prepare the Isobiotec cell-breaker, assemble it by inserting the ball with the size of your choosing inside the machine and screw the lids on tightly. For PBMCs 12 μm gap is effective. Place on aluminium foil TemperatureOn ice and push Amount3 mL of KPBS through the machine to wash it. Carefully tap dry.
Note
Note: There will be residual KPBS left in the cell-breaker (approximately Amount200 µL ): this is optimal.


Transfer the PBMCs from Step 20 into a KPBS rinsed, ice-cold Isobiotec cell-breaker with gap-size of 12 μm using a 1-ml syringe and 21G needle. Homogenise the cells with 15-20 passes through the cell breaker using two 1-ml syringes.
Note
Note: The homogenisation requires more force with more passes. Pay extra care to make sure the syringes are securely in their seals and that the sample doesn’t leak out. If you encounter too much pressure for passing the homogenate through the cell-breaker, consider using a ball that leaves a larger gap.

Collect the homogenate from the cell breaker into a fresh Eppendorf tube using a 1-ml syringe.
Note
Note: To extract as much sample as possible from the cell-breaker post-homogenisation, push air into the cell-breaker using a syringe and collect from the other seal using another syringe.

Centrifuge at Centrifigation1500 x g, 4°C, 00:02:00 .
Centrifigation
Transfer the supernatant to a fresh Eppendorf tube TemperatureOn ice .
Note
Note: The pellet contains fragments of plasma membrane and the nucleus and non-homogenised cells.


Add the supernatant from Step 26 to Amount100 µL of the prewashed beads (from Step 18). Mix by pipetting gently three times, then place on a belly-dancer orbiter for Duration00:05:00 at Temperature4 °C .
Note
Note: Make sure the homogenate/bead slurry is in constant motion.

Pipetting
Mix

Note
The following steps would optimally be performed in a Temperature4 °C cold room. If not available, then keep working TemperatureOn ice .

Place the IPs from Step 27 on a Dyna-Mag tube holder for Duration00:00:30 to immobilise the beads. Discard the supernatant or collect as flowthrough sample.
Note
Note: The flowthrough now contains cell debris and other organelles, and any lysosomes that remained unbound to the beads.


Resuspend the beads from Step 28 in Amount1 mL of KBPS and immobilise the beads by placing the tubes in a Dyna-Mag tube holder for Duration00:00:30 . Discard the supernatant.

Repeat Step 29: Resuspend the beads in Amount1 mL of KBPS and immobilise the beads by placing the tubes in a Dyna-Mag tube holder for Duration00:00:30 . Discard the supernatant.
Resuspend the beads in Amount1 mL of KPBS and transfer to a new Eppendorf tube TemperatureOn ice .

Place the tubes in a Dyna-Mag tube holder for Duration00:00:30 . Discard the supernatant.

The lysosomes attached to the dry beads from Step 32 can now be either eluted off the beads with SDS or Triton-X100 based lysis buffer or stored as they are at Temperature-80 °C for later use. Alternatively, they can be immediately processed for:
  1. Immunoblotting analysis
  2. Lipidomic analysis
  3. Metabolomic analysis
  4. Proteomic analysis


Note
The following steps instruct how to wash the Isobiotec cell-breaker between samples and at the end of the experiment.
Open the cell-breaker from one side.
Take the metal ball out and rinse with MillIQ-water.
Flush the cell breaker thoroughly with MilliQ-water.
Note
Note: Use 5-ml syringes filled with MilliQ-water to flush through both of the syringe inlets whilst covering the opening in the side of the cell breaker from Step 34.

Reassemble the cell-breaker by re-inserting the metal ball into the instrument and close the side panel tightly using the screws.
Flush the cell breaker through the syringe inlets with Amount5 mL of KPBS for both inlets using 5-ml syringes.
Note
Note: There will be residual KPBS left in the cell-breaker (approximately Amount200 µL ), this is optimal.



Proceed to homogenise the next sample.
Once done using the homogeniser for the experiment, repeat Step 36.
Take all pieces apart (both side panels, panel screws and the metal ball).
Clean each part with generous amount of 70% (v/v) ethanol in MilliQ-water.
Wipe all parts dry and leave pieces apart to air-dry DurationOvernight .
Note
Note: Packing up the cell-breaker before it is dry will lead to development of rust and colouring of the metal parts.


Overnight