May 10, 2024

Public workspaceFlowcytometry analysis of lysosomal pulldown with anti-TMEM192 magnetic beads from PBMCs

DOI
dx.doi.org/10.17504/protocols.io.n2bvj378xlk5/v1
Flowcytometry analysis of lysosomal pulldown with anti-TMEM192 magnetic beads from PBMCs
  • 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Enrico Bagnoli
Open access
DOI: dx.doi.org/10.17504/protocols.io.n2bvj378xlk5/v1
Protocol Citation: Enrico Bagnoli, Karolina Zeneviciute, Esther Sammler, Dario R Alessi 2024. Flowcytometry analysis of lysosomal pulldown with anti-TMEM192 magnetic beads from PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj378xlk5/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: February 13, 2024
Last Modified: May 10, 2024
Protocol Integer ID: 95163
Keywords: Organelle-IP, Lyso-IP, Flowcytometry, PBMCs, TMEM192, Immunoprecipitation
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
Abstract
We describe a method that allows the staining of intact lysosomes using lysotracker for flow cytometry analysis. The analysis can provide a quantitative indication of the level of enrichment of the organelle immunoprecipitation (% of lysososomal+ beads) and can be used as a quality control check for the purity of the immunoprecipitated lysoIPs. Since only a small fraction of the immunoprecipitated material is needed for flow cytometry, this technique can be coupled with other assays, like western blotting or proteomics. While this protocol is focused on the staining and detection of lysosomes, it can be easily modified to analyse different organelles by changing the staining procedure and adjusting the parameters of the cytometer.
Intro
Intro
The following protocol allows the detection and analysis of the lysosomes isolated by immunoprecipitation with anti-TMEM192 magnetic beads from peripheral blood mononuclear cells (PBMCs). Cells were incubated with or without the lysotracker prior to the lysosomal immunoprecipitation. Additionally, PBMCs were treated with the V-ATPase inhibitor Bafilomycin A1, that blocks the acidification of the lysosomes and drastically reduces the signal. We included this step as a further proof of the functionality of the isolated lysosomes, but it can be avoided if unnecessary.
This protocol can be adapted to any other cell type.

Note
We noticed that TMEM192 beads have a strong autofluorescence signal, particularly in the FITC and mCherry channels. Using a higher excitation wavelength solved this issue. Specifically, we used the Deep Red Lysotracker (max ex 647, max em 668) and used the APC channel for flow cytometry analysis.

Materials and reagents
Materials and reagents

  • PBMCs (or other cells)
  • Bafilomycin A1 (Enzo LifeScience, BML-CM110-0100)
  • Deep Red Lysotracker (Thermo, L12492)
  • DMSO
  • anti-TMEM192 magnetic beads
  • BSA magnetic beads
  • KPBS
  • Flow cytometry tubes
  • Flow cytometer with APC lasers and filters
  • Water bath
PBMCs extraction
PBMCs extraction
  • Extract PBMCs as outlined in the protocol
https://www.protocols.io/view/purification-of-tag-less-lysosomes-from-cells-isol-b5ziq74e (steps1-11)

  • Divide PBMCs into different tubes, according to the experimental design.
Note
We divided cells from each donor into 4 tubes.
Tube 1: - Baf, - Tracker
Tube 2: - Baf, + Tracker
Tube 3: + Baf, - Tracker
Tube 4: + Baf, + Tracker
  • Leave PBMCs in water bath at Temperature37 °C

Bafilomycin A1 treatment
Bafilomycin A1 treatment

  • Prepare a Concentration100 micromolar (µM) working stock of Bafilomycin A1 in DMSO.
  • Add Bafilomycin to tube 3 and 4 to a final concentration of Concentration200 nanomolar (nM)
  • Add the same volume of DMSO to tube 1 and 2
  • Incubate at Temperature37 °C for Duration04:00:00 in the waterbath, gently mixing cells every 20min to avoid cell attachment

Note
Duration and concentration of the Bafilomycin A1 treatment can be tailored according to the specific requirements


4h
Lysotracker staining
Lysotracker staining

  • Prepare Concentration1 millimolar (mM) stock of Lysotracker in DMSO, if not already prepared
  • Make 2 μM solution (Amount2 µL l of Stock in Amount1000 µL of buffer)
  • Add dye to a final concentration of Concentration50 nanomolar (nM) (1:40 dilution of 2 μM working solution), into Tube 2 and Tube 4, mix gently and incubate Temperature37 °C Duration00:30:00
  • Add the corresponding volume of DMSO into Tube 1 and 3.

30m
Immunoprecipitation
Immunoprecipitation
Note
The IP is normally performed using 50 μl of beads/sample. However, only a small fraction of the beads is analysed (approx 1/200 of initial beads), the rest of the IP can be used for WB or other techniques. The initial amount of beads can also be scaled down.
  • At the end of the IPs, for each of the initial tubes, there should be 2 samples, a TMEM192 IP and a control IP (BSA-beads).
  • Add Amount400 µL of KPBS to each FACS tube and label them accordingly

Flow cytometry and analysis
Flow cytometry and analysis
  • Turn on the cytometer.
  • Prepare windows and gates according to the lysotracker used. LysoTracker™ Deep Red has the excitation at 647nm and emission at 668nm (APC channel)
  • Dilute the samples. We used a dilution of 1/10, by adding Amount40 µL of stained beads to Amount400 µL of KPBS into FACS tube.
  • Run the samples on a flow cytometer.

Note
On a BD LSR Fortessa we used the following voltages:
FSC: 600
SSC: 240
APC: 700




FlowJo analysis of FCS files
  • Isolate the beads from the debris by gating on the SSC vs FSC plot.
  • Isolate positive beads either on the APC histograms or on the APC vs FSC plot
Note
Use the appropriate control to gate for the positive beads (TMEM192 pulldown without tracker)
Protocol references