Flowcytometry analysis of lysosomal pulldown with anti-TMEM192 magnetic beads from PBMCs
Dario R Alessi, Enrico Bagnoli, Esther Sammler, Karolina Zeneviciute
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.
Steps
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.
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
Extract PBMCs as outlined in the protocolhttps://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.
* Leave PBMCs in water bath atNote
We divided cells from each donor into 4 tubes. Tube 1: - Baf, - TrackerTube 2: - Baf, + TrackerTube 3: + Baf, - TrackerTube 4: + Baf, + Tracker 37°C
Bafilomycin A1 treatment
- Prepare a
100micromolar (µM)working stock of Bafilomycin A1 in DMSO. - Add Bafilomycin to tube 3 and 4 to a final concentration of
200nanomolar (nM) - Add the same volume of DMSO to tube 1 and 2
- Incubate at
37°Cfor4h 0m 0sin the waterbath, gently mixing cells every 20min to avoid cell attachment
Lysotracker staining
- Prepare
1millimolar (mM)stock of Lysotracker in DMSO, if not already prepared - Make 2 μM solution (
2µLl of Stock in1000µLof buffer) - Add dye to a final concentration of
50nanomolar (nM)(1:40 dilution of 2 μM working solution), into Tube 2 and Tube 4, mix gently and incubate37°C0h 30m 0s - Add the corresponding volume of DMSO into Tube 1 and 3.
Immunoprecipitation
Perform endogenous organelle IP as in dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1 (steps 12-33)
- Add
400µLof KPBS to each FACS tube and label them accordingly
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
40µLof stained beads to400µLof KPBS into FACS tube. - Run the samples on a flow cytometer.
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)
