TITLE: Staining of cells with GolgiTracker for Golgi flow cytometry analysis
AUTHORS: Enrico  Bagnoli, Rotimi  Fasimoye, Dario R Alessi

[DESCRIPTION]
We describe a method that allows the staining of intact Golgi using GolgiTracker for subsequent flow cytometry analysis. The analysis can provide a quantitative indication of the level of enrichment of the organelle immunoprecipitation (% of GOLGI+ beads) and can be used as a quality control check for the purity of the immunoprecipitated Golgi. 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.
The staining can be performed directly on the cells, or, alternatively, after the Golgi has been isolated from the cells (For our Golgi immunoprecipitation protocol, see dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1). In our experience, the first method (which is described in this protocol) results in a cleaner signal. It should be noted that the staining is performed in KPBS, the buffer is used as optimal for organelles isolation. This buffer is compatible with the GolgiTracker staining, however other staining, particularly those using antibodies, might require a buffer containing Mg2+ or Ca2+. While this protocol is focused on the staining and detection of Golgi, it can be easily modified to analyse different organelles by changing the staining procedure and adjusting the parameters of the cytometer.


[GUIDELINES]
For a detailed method on how to introduce GolgiTAG to cells and immunoprecipitate intact Golgi, see dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1.

[STEPS]
SECTION: Flow cytometry
5. Flow cytometry analysis
 

SECTION: Staining on cells + Immunoprecipitation
2. Staining of cultured cells with GolgiTracker 
SECTION: Staining on cells + Immunoprecipitation
3. Isolation of intact Golgi using GolgiTag immunoprecipitation method


SECTION: Materials and reagents
1. 
Cell culture
SECTION: Analysis
6. Data analysis using FlowJo.
 

SECTION: Materials and reagents
1.1. Grow HEK293 cells stably expressing GolgiTag or ControlTag (generated as described in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1) in 10cm2 Petri dish until near confluent.

Note: For each experimental conditions, two plates will be needed: one to be stained with GolgiTracker, and a control plate (not stained with GolgiTracker).
SECTION: Staining on cells + Immunoprecipitation
2.2. Add the GolgiTracker to the cells to a final concentration of 5 micromolar (µM) (i.e. 1:100 dilution). Add DMSO vehicle (1% v/v) to control cells.
 

SECTION: Staining on cells + Immunoprecipitation
2.1. Prepare a 500 micromolar (µM) stock of GolgiTracker in DMSO and dissolve thoroughly by vortexing.
SECTION: Staining on cells + Immunoprecipitation
2.4. 
Discard media and add fresh media and incubate for further 30 minat 37 °C
SECTION: Staining on cells + Immunoprecipitation
2.3. 
Incubate at 37 °C for 30 min
SECTION: Staining on cells + Immunoprecipitation
3.1. Perform Golgi immunoprecipitation as described in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1

SECTION: Staining on cells + Immunoprecipitation
3.2. At the end (Step 56 in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1), add  500 µL to the beads (instead of adding lysis buffer) and gently resuspend by pipetting up and down.
SECTION: Flow cytometry
5.2. Turn on machine following relative SOPs. If first user of the day, also perform the starting procedure.
SECTION: Flow cytometry
5.3. Prepare working windows and gates. The tracker used can be analysed in the FITC channel.
 

SECTION: Flow cytometry
5.5. When FSC, SSC and FITC voltages are fine, start running the samples.
For each sample, record at least 20k events, but if possible 50k is a better option.
SECTION: Flow cytometry
4. Preparation of samples for flow cytometry.
SECTION: Flow cytometry
4.2. Dilute samples. We used a dilution of 1:100 (or 1:200)  from the final IPs. So from the500 µL take 40 µLand resuspend in a FACS tube with 400 µL on on ice 

 

SECTION: Flow cytometry
5.4. If the samples are run for the first time, adjust voltages for the Forward and Side scatter and the FITC channel. Otherwise use the parameters already optimized before.
 

SECTION: Flow cytometry
5.6. At the end, clean the machine and save the data (FCS 3.0 files)
SECTION: Analysis
6.2. Gate the beads from the SSC and FSC plot as shown in the figure below.


 

 

SECTION: Analysis
6.3. From these population calculate the % of GolgiTracker+ beads either from the FITC vs FSC plot or from the FITC histogram. 
 

SECTION: Analysis
6.4. In the workspace, create tables and graphs of the relevant samples.

SECTION: Analysis
6.6. Perform statistical analysis using Prism software or equivalent.


SECTION: Flow cytometry
4.1. Prepare and label the appropriate amount of FACS tube. For example:
GolgiTAG +Tracker
GolgiTAG  -Tracker
HA-Empty +Tracker
HA-Empty  -Tracker
SECTION: Flow cytometry
5.1. Check the waste container of the cytometer is not full. Check the buffer container of the cytometer is not empty. Refill or empty accordingly.
SECTION: Analysis
6.1. Open the software and drag the samples to be analyzed in the main window
SECTION: Analysis
6.5. Export tables and images in the preferred format.