Multicolor flow cytometric analysis of regulatory T cells from mouse spleen

Notes:

• Always prepare reagents freshly.

PEB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with autoMACS®. Rinsing Solution (# 130-091-222). Keep buffer cold (2–8 °C). Always use freshly prepared buffer. Do not use autoMACS. Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.

Let the vials warm up to room temperature to avoid water condensation. Reconstitute one vial of lyophilized Viobility Fixable Dye by adding 100 μL anhydrous DMSO to the dye vial and mix until fully dissolved. Aliquot the solution and store at –20 °C under anhydrous conditions (desiccant) and protected from light for up to 1 month.

Notes:

• The required total buffer volumes should be calculated beforehand; volumes will depend on the number of cells to be analyzed as well as the number of tests to be performed (for more information, please refer to the Anti-FoxP3 antibodies data sheet).

1. Prepare Fixation/Permeabilization Solution by diluting 1:4 the Fixation/Permeabilization Solution 1 with the Fixation/Permeabilization Solution 2, (i.e. for 10⁶ cells use 0.25 mL of Fixation/Permeabilization Solution 1 plus 0.75 mL of Fixation/Permeabilization Solution 2).
2. Prepare a 1× solution of Permeabilization Buffer by diluting the 10× Permeabilization Buffer with deionized or distilled water. 
   ▲ Note: Before dilution make sure that buffer does not contain any precipitates.

Notes: 

• For details on the use of the gentleMACS™ Dissociators, refer to the gentleMACS Dissociator user manuals. 
• To obtain the results shown in this protocol one spleen per C Tube has been dissociated. 

1. Transfer mouse spleen into the gentleMACS C Tube containing the following amount of PEB buffer: 
   1–2 mouse spleens: 3 mL 
   3–4 mouse spleens: 6 mL 
   5–6 mouse spleens: 9 mL 
2. Tightly close C Tube and attach it upside down onto the sleeve of the gentleMACS Dissociator.  
   ▲ Note: Close C Tube tightly beyond the first resistance.  
   ▲ Note: Ensure that the sample material is located in the area of the rotor/stator. 
   
3. Choose and run one of the following gentleMACS Programs: 
       1–2 mouse spleens: m_spleen_01 
       3–6 mouse spleens: m_spleen_04 
   
4. After termination of the program, detach C Tube from the gentleMACS Dissociator. 
   
5. (Optional) Perform a short centrifugation step to collect the sample material at the bottom of the tube. 
   
6. Resuspend sample and apply the cell suspension to a MACS SmartStrainer (70 μm), placed on a 15 mL tube (1–2 mouse spleens per C Tube) or to a 50 mL tube (3–6 mouse spleens per C Tube). 
   ▲ Note: Moisten MACS SmartStrainer with 2 mL buffer before use. 
   ▲ Note: Dissociated tissue can be removed from the closed C Tube by pipetting through the septum-sealed opening in the center of the cap of the C Tube. Use ART® 1000 REACH™ 1000 μL pipette tips. 
   
7. Wash the filter with 5 mL of PEB buffer. 
   
8. Discard the filter and centrifuge cell suspension at 300×g for 10 minutes at room temperature. Aspirate supernatant completely. 
   
9. Resuspend cells in PEB buffer to an appropriate volume and proceed to flow cytometry staining. 

Notes:

• Volumes given below are for up to 10⁷ nucleated cells. When working with fewer than 10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁷ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

1. Determine cell number.
2. Wash cells in 1 mL of 1× PBS and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cells in 100 μL of 1× PBS.
4. Add 1 µL of Viobility™ 405/520 Fixable Dye.
5. Mix well and incubate for 15 minutes in the dark at room temperature.
   ▲ Note: Higher temperatures and/or longer incubation times may lead to nonspecific cell labeling. Working on ice requires increased incubation times.
6. Wash cells by adding 1 mL PEB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
7. (Optional) Repeat step 6.
8. Proceed with surface and intracellular staining.

Notes:

• To obtain the results shown in this protocol a total of 1×10⁶ splenocytes has been stained in a total volume of 100 µL.

1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Prepare a master mix with all fluorochrome-conjugated antibodies according to the respective data sheets.
4. Add master mix to the cell suspension.
5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
6. Wash cells by adding 1 mL of PEB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
7. Proceed with intracellular staining.

Notes:

• Always prepare reagents freshly. Failure to do so may lead to sub-optimal results.

1. Resuspend cells in 1 mL of cold, freshly prepared Fixation/Permeabilization Solution.
2. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C).
3. Wash cells by adding 1 mL of cold buffer per 10 cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
4. Wash cells by adding 1 mL of cold 1× Permeabilization Buffer and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
5. Resuspend cells in 1× Permeabilization Buffer and add the corresponding volume of the Anti-FoxP3 antibody according to the data sheet.
6. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C).
7. Wash cells by adding 1 mL of cold 1× Permeabilization Buffer and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
8. Resuspend cells in an appropriate volume of 1× Permeabilization Buffer Buffer and proceed to flow cytometry analysis.

Notes:

• Prior acquisition of samples in a flow cytometer, e.g., the MACSQuant® Analyzer 10, make sure lasers are properly calibrated (by using, e.g., the MACSQuant Calibration Beads), and compensation of the spillover of fluorochrome emissions has been established in advance using cells or the MACS® Comp Bead Kit, anti-REA. Prior acquisition adjust also forward scatter (FSC)/side scatter (SSC) parameters to properly locate all leukocyte populations in the plots.

1. Exclude doublets by using the FSC-H/FSC-A parameters.
2. Use gated single cells to exclude erythrocytes and debris using the SSC/FSC parameters.
3. Use the SSC/FSC gate to exclude dead cells from the analysis using the Viobility Dye/FSC.
4. Use gated Viobility™ Dye–negative (viable) cells to identify regulatory T cells.

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