Application protocol

Multicolor flow cytometric analysis of monocytes from mouse spleen

This application protocol describes the flow cytometric analysis of monocytes after spleen dissociation from healthy C57BL/6 mice. Viable single cells from mouse spleens are easily obtained using the gentleMACS™ Technology. For downstream flow cytometric analysis of monocytes, we have designed a validated multicolor flow cytometry panel, using our REAfinity™ Recombinant Antibodies and Viobility™ Fixable Dyes. In addition, we provide an optimized gating strategy for the analysis of monocytes.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only. 

For dissociation of mouse spleen

  • Phosphate-buffered saline (PBS), pH 7.2
  • PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
  • Spleen Dissociation Kit, mouse (# 130-095-926)
  • gentleMACS Dissociator™ (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • MACS® SmartStrainers (70 µm) (# 130-098-462)
  • (Optional) MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubator at 37 °C
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.

For flow cytometry staining and analysis

  • Phosphate-buffered saline (PBS), pH 7.2
  • Viobility™ 405/520 Fixable Dye (# 130-110-206, # 130-109-814)
  • PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
  • CD11b-VioBlue® (# 130-113-810)
  • CD3ε-PE-Vio® 615 (# 130-109-245)
  • CD19-PE-Vio 615 (# 130-112-042)
  • CD335 (NKp46)-PE-Vio 615 (# 130-112-364)
  • CD45-APC-Vio 770 (# 130-110-800) 
  • Anti-F4/80-FITC (# 130-117-509)
  • CD192 (CCR2)-PE (# 130-117-548)
  • Anti-Ly-6C-PE-Vio 770 (# 130-111-918)
  • Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
  • (Optional) MACSQuant Calibration Beads (# 130-093-607)
  • (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
Matching products:

Protocol


View timeline

This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

Notes:

  • Always prepare reagents freshly.

Preparation of the PEB buffer

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.

Reconstitution of the Viobility™ 405/520 Fixable Dye

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:

  • For details on the use of the gentleMACS™ Dissociators, refer to the gentleMACS Dissociator user manuals.
  • For information about buffer preparation and enzyme reconstitution refer to the Spleen Dissociation Kit data sheet.
  • To obtain the results shown in this protocol one spleen per C Tube has been dissociated.
  1. Prepare enzyme mix of the Spleen Dissociation Kit by adding 2.4 mL of 1× Buffer S, 50 µL of Enzyme D, and 15 µL of Enzyme A into a gentleMACS C Tube.
  2. Transfer one mouse spleen into the gentleMACS C Tube containing the enzyme mix
  3. 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.
  4. Run the gentleMACS Program m_spleen_02. If using the heating function of the gentleMACS Octo Dissociator with Heaters run program 37C_m_SDK_1 and continue with step 9.
  5. After termination of the program, detach C Tube from the gentleMACS Dissociator.
    Note: The spleen will not be completely dissociated after this step. In the unexpected event that the spleen is not dissociated at all, repeat steps 4 and 5.
  6. Incubate sample for 15 minutes at 37 °C under slow continuous rotation using the MACSmix™ Tube Rotator.
  7. Attach C Tube upside down onto the sleeve of the gentleMACS Dissociator. 
    Note: Ensure that the sample material is located in the area of the rotor/stator.
  8. Run the gentleMACS Program m_spleen_03.
  9. After termination of the program, detach C Tube from the gentleMACS Dissociator.
  10. (Optional) Perform a short centrifugation step to collect the sample material at the bottom of the tube.
  11. Resuspend sample and apply the cell suspension to a MACS SmartStrainer (70 μm), placed on a 15 mL 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.
  12. Wash the filter with 2.5 mL of 1× Buffer S.
  13. Discard the filter and centrifuge cell suspension at 300×g for 10 minutes at room temperature. Aspirate supernatant completely.
  14. Resuspend cells in PEB buffer to the required volume for flow cytometry staining.

Configuration of the panel 

LaserAntibody specificityFlurochromeClone
VioletCD11bVioBlue® REA592
 Viobility™  405/520 Fixable Dye 
BlueAnti-F4/80FITCREA126
CD192 (CCR2)PEREA538
CD3ε*PE-Vio® 615REA606
CD19*PE-Vio 615REA747
CD335 (NKp46)*PE-Vio 615REA815
Anti-Ly-6CPE-Vio 770REA796
RedCD45APC-Vio 770REA737
* Exclusion channel containing lineage markers to exclude T cells (CD3ε), B cells (CD19), and NK cells (CD335 (NKp46)) from the analysis.

Labeling of cells with Viobility™ 405/520 Fixable Dye

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 non-specific 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. Resuspend cells in an appropriate volume of PEB buffer and proceed  with surface staining.

Surface 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. Resuspend cells in an appropriate volume of PEB 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 monocytes.
View details

Gating strategy showing the analysis of monocytes from healthy mouse spleen. Splenocytes from C57BL/6 mice were stained using the described panel. Monocytes lack expression of the F4/80 molecule as well as T, B, and NK cell lineage markers (CD3ε, CD19, CD335 (NKp46)), while express high levels of the CD11b and Ly-6C molecules. In addition, the expression of the chemokine receptor CD192 (CCR2) helps to identify monocytes within mouse spleen.

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