TITLE: Standardised flow cytometric protocol for the detection of immune cells subsets in breast cancer patients. 
AUTHORS: Nicole Chicken, Pieter Meyer, Ronald Anderson, Bernardo Rapoport, Catherine Worsley

[DESCRIPTION]
The tumour microenvironment supports tumour growth in several ways including manipulating the immune response to cancer cells. Developing accurate and reproducible assays to detect immune and characterize immune cells in cancer patients is important for understanding their impact on tumour growth and development. Multicolour flow cytometry allows for characterization of many cell populations in a single sample. In this protocol, we optimized detecting several cytotoxic and suppressive immune cell subsets that may impact on patient outcomes. In addition, this protocol can be used to monitor changes in immune cells during and after chemotherapy.




[BEFORE_START]
30 minutes before running a sample, allow the following to come to room temperature: 
-1x Phosphate Buffered Saline (PBS)
-Fetal Bovine Serum (FBS) (ThermoFisher Scientific, Massachussetts, USA)
-Brilliant Violet stain buffer (BD Biosciences, New Jersey, USA.563794)

Note:
- Samples must be centrifuged with the brake on and at room temperature.
-Samples must be incubated at room temperature. 
- The reagent tubes, liquid antibodies, and other light-sensitive reagents must be kept in the dark as much as possible. 

[STEPS]
SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
23. Label a DURAClone IM Phenotyping Treg kit tube 1, and add 5 µL to the tube.

SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
24. Add 50 µL to reagent tube 1, and vortex for 10 seconds. Incubate the tube in the dark for 15 minutes at room temperature (RT). 

SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
25. Add 3 mL to reagent tube 1 and centrifuge the tube at 500 xg for 5 minutes.

SECTION: Post-acquisition
41. Once all events have been acquired, copy all work to an external storage device by selecting 'Export FCS files' from the 'File' menu, choosing the tubes you want to save, selecting your desired file path and selecting 'Ok'.
SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
26. Discard the supernatant, and gently vortex the cell pellet for 8 seconds.
SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
27. Resuspend the cell pellet in 50 µL

SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
28. Add 5 µL and vortex for 8 seconds. Incubate the tube in the dark for 15 minutes at RT.

SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
29. Add 400 µL and vortex for 8 seconds. 

SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
30. Label a DURAClone IM Phenotypic Treg kit Tube 2, and transfer the contents of reagent tube 1 into reagent tube 2 and vortex for 10 seconds.
SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
31. Incubate the tube in the dark for 60 minutes at RT.

SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
32. Add 3 mL and incubate in the dark for 15 minutes at RT. 

SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
33. Centrifuge the tube at 500 xg for 5 minutes. Discard the supernatant and vortex the pellet for 8 seconds.
SECTION: DURAClone IM Phenotyping Treg kit test protocol for 1 sample2
34. Resuspend the cell pellet in 3 mL and centrifuge the tube at 500 xg for 5 minutes. Discard the supernatant and cortex the pellet for 8 seconds. 

35. Resuspend the pellet in 500 µL and acquire the results on the flow cytometer. Run a daily clean before running any DURAClone IM Phenotyping basic kit tubes. 

SECTION: DURAClone IM Phenotyping Basic kit protocol for 1 sample3
36. Label a DURAClone IM Phenotyping Basic kit reagent tube, add the following, and then vortex for 8 seconds: 
5 µL 
5 µL 

SECTION: DURAClone IM Phenotyping Basic kit protocol for 1 sample3
37. Add 30 µL then 100 µL into the reagent tube, and vortex for 10 seconds. Incubate the tube in the dark for 15 minutes at RT. 

SECTION: DURAClone IM Phenotyping Basic kit protocol for 1 sample3
38. Add 2 mL and vortex for 3 seconds. Incubate the tube in the dark for 15 minutes at RT.

SECTION: DURAClone IM Phenotyping Basic kit protocol for 1 sample3
39. Centrifuge the tube at 200 xg for 5 minutes. Discard the supernatant, and resuspend the cell pellet in 3 mL . 

SECTION: DURAClone IM Phenotyping Basic kit protocol for 1 sample3
40. Centrifuge the tube at 200 xg for 5 minutes, discard the supernatant, and resuspend the cell pellet in 500 µL . Acquire the results on the flow cytometer. 

SECTION: Flow cytometer start-up and daily quality control1
1. Refill the sheath tank, empty the waste tank, and check that there is enough cleaning solution in the internal compartment of the flow cytometer. 
SECTION: Flow cytometer start-up and daily quality control1
2. Turn on the CytoFLEX flow cytometer and the PC and allow both to start up. Log in to the PC and open the CytExpert software. 
SECTION: Flow cytometer start-up and daily quality control1
3. Initiate the CytoFLEX daily start-up program and load a tube of deionised water when prompted. 
SECTION: Flow cytometer start-up and daily quality control1
4. To run daily Quality Control (QC), make sure that the lot-specific target value file for the CytoFLEX Ready to Use Daily QC fluorophores are loaded onto the CytExpert software.     
SECTION: Flow cytometer start-up and daily quality control1
5. Open the QC/Standardisation tab and select the CytoFLEX Ready to Use Daily QC fluorophores lot number from the relevant drop-down menu. Vortex the bottle of QC fluorophores for 10 seconds before adding 10 drops into a clean 5mL tube or 34 drops into one well of a 96-well plate. Put the tube/plate into the sample loader and initialise the instrument, then select start. 
SECTION: Flow cytometer start-up and daily quality control1
6. If the QC step fails, try vortexing the aliqoted QC fluorphores and re-running the QC/standardisation step. If it fails again, run a daily clean and prime and re-run. If it fails again, contact Beckman Coulter.  
SECTION: Flow cytometer start-up and daily quality control1
7. Do a 3 minute FlowClean 3 minute deionised water Daily Clean before proceeding. 
SECTION: Compensation set-up1
8. First prepare the required unstained and single color controls using compensation beads or cells. Then select New Compensation under 'File" and save. Select the channels you wish to run compensation for, select sample type and uncheck tubes not needed for the experiment. Select 'Ok'. The CytExpert software will create a list of matching empty tubes in the Tube panel as well as all required plots. 
SECTION: Compensation set-up1
9. Load the correct unstained or single color sample fluorospheres or cells and select 'Run'
in the Acquisition panel. Adjust the scatter gate if necessary. Use the slider scales so that the positive and negative peaks are suitably positioned, and adjust the positive and negative gates accordingly. Select 'Record'. Repeat for all tubes. 
SECTION: Compensation set-up1
10. After running all tubes, generate the compensation matrix by selecting 'Compensation Calculation' from the toolbox or settings menu. Select 'Save As' to export
the matrix as a .comp file and to save the values to the compensation library, select 'Save to Compensation Library' . Select 'Ok' and 'Close'.  
SECTION: Compensation set-up1
11. To save or overwrite the compensation matrix to the library, select 'Open compensation experiment' and run the compensation controls. Once completed, calculate compensation. Then save the compensation matrix to the compensation library. Run a 3 minute Flow Clean 3 minute deionised water daily clean before proceeding.
SECTION: Setting up the gating strategy for the Treg kit2
12. Create an analysis protocol on the CytExpert software and insert a series of dual parameter plots. Make sure to set the discriminator on the forward scatter (FS) parameter/axis low enough to prevent the exclusion of any lymphocytes from the data acquisition. 
 
SECTION: Setting up the gating strategy for the Treg kit2
13. Create a CD45-Kr0 vs. Side scatter (SSC) dot plot and apply the leukocyte gate. Next, create a gate around the CD45+ lymphocytes. (Figure 1)
SECTION: Setting up the gating strategy for the Treg kit2
14. Create a CD4-PC7 vs. CD3-AA750 dot plot and gate the CD3+CD4+T-cells. (Figure 2)
SECTION: Setting up the gating strategy for the Treg kit2
15. Create the following dot plots, and apply the CD3+CD4+ gate to each: 
- A CD4-PC7 vs. CD25-PE dot plot. Gate the CD4+CD25+ population.  (Figure 3)
-A CD4-PC7 vs. FoxP3-A647 dot plot. Gate the CD4+FoxP3+ population. (Figure 4)
-A CD4-PC7 vs. Helios-PBE dot plot. Gate the CD4+Helios+ population. (Figure 5)
-A CD4-PC7 vs. CD39-PC5.5 dot plot. Gate the CD4+CD39+ population. (Figure 6)
-A CD4-PC7 vs. CD45RA-FITC dot plot. Gate the CD4+CD45RA+ population. (Figure 7)
-A FoxP3-A647 vs. Helios-PBE dot plot. Gate the FoxP3+Helios+ population. (Figure 8)
SECTION: Setting up the gating strategy for the Basic kit3
16. Create an analysis protocol on the CytExpert software and insert a series of dual parameter plots. Make sure to set the discriminator on the forward scatter (FS) parameter/axis low enough to prevent the exclusion of any lymphocytes from the data acquisition. 
 
SECTION: Setting up the gating strategy for the Basic kit3
17. Create a CD45-KrO vs. side scatter (SSC) dot plot and create a gate around the CD45+ lymphocytes.
SECTION: Setting up the gating strategy for the Basic kit3
18. Create three plots as outlined below: 
- A CD14-PC7 vs. SSC dot plot. Gate the CD14+ cells (monocytes). 
-A CD19-ECD vs. D3-APC-A750 dot plot. Gate the CD19+ cells (B-cells).
-A CD56-PE vs. CD3-APC-A750 dot plot. Create and apply a Boolean gate "Lymphocytes AND (NOT CD19+)". Gate the CD56+ (Natural Killer (NK) cell) and CD3+ (CD3+ T-cells) cell populations. 
SECTION: Setting up the gating strategy for the Basic kit3
19. Create a CD56-PE vs. CD16-FITC dot plot. Apply the gate from the CD56-PE vs. CD3-APC-A750 dot plot. Gate the CD56+ high NK cells.
SECTION: Setting up the gating strategy for the Basic kit3
20. Create a CD16-FITC vs. CD14-PC7 dot plot. Apply the CD14+ gate to this plot, and create separate gates around each of the following populations:   
- CD14+ high CD16- monocytes
-CD14+ high CD16+ monocytes
-CD14+CD16+ high monocytes 
SECTION: Setting up the gating strategy for the Basic kit3
21. Create a CD4-APC vs. CD8-A700 dot plot.  Apply the CD3+ T-cells gate from the CD56-PE vs. CD3-AA750 dot plot. Gate the CD4+ T-cells, and then the CD8+ T-cells. 
SECTION: Setting up the gating strategy for the Basic kit3
22. Record the mean fluorescence intensity (MFI) of all gated populations. 
SECTION: Post-acquisition
42. Run a daily clean, close CytExpert, and shut down the PC and flow cytometer.