TITLE: Flowcytometry analysis of lysosomal pulldown with anti-TMEM192 magnetic beads from PBMCs
AUTHORS: Enrico Bagnoli, Karolina Zeneviciute, Esther Sammler, Dario R Alessi

[DESCRIPTION]
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]
SECTION: Materials and reagents
2. 
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
SECTION: Intro
1. 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.

 

SECTION: Immunoprecipitation
6. Perform endogenous organelle IP as in dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1 (steps 12-33)
 
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 400 µL of KPBS to each FACS tube and label them accordingly

SECTION: Lysotracker staining
5. 
Prepare 1 millimolar (mM) stock of Lysotracker in DMSO, if not already prepared
Make 2 μM solution (2 µLl of Stock in 1000 µLof buffer)
Add dye to a final concentration of  50 nanomolar (nM) (1:40 dilution of 2 μM working solution), into Tube 2 and Tube 4, mix gently and incubate  37 °C 30 min
Add the corresponding volume of DMSO into Tube 1 and 3.

SECTION: Flow cytometry and analysis
7. 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 40 µL of stained beads to 400 µL of KPBS into FACS tube.
Run the samples on a flow cytometer.

 




SECTION: Flow cytometry and analysis
7.1. 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
 
 
SECTION: PBMCs extraction
3. 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.
 
Leave PBMCs in water bath at 37 °C 

SECTION: Bafilomycin A1 treatment
4. 
Prepare a  100 micromolar (µM) working stock of Bafilomycin A1 in DMSO.
Add Bafilomycin to tube 3 and 4 to a final concentration of 200 nanomolar (nM) 
Add the same volume of DMSO to tube 1 and 2
Incubate at 37 °C for 240 min in the waterbath, gently mixing cells every 20min to avoid cell attachment