MBTP 14: Immunophenotyping of macrophages from mouse spleen using flow cytometry
Miltenyi Biotec-tested panel 14 (MBTP 14)
Miltenyi Biotec-tested panel 14 (MBTP 14)
This application protocol describes the flow cytometric analysis of macrophages after spleen dissociation from healthy C57BL/6 mice. Mouse spleens are easily dissociated using the gentleMACS™ Dissociators to quickly obtain viable single cell suspensions ready for flow cytometric analysis.
Protocol
Labeling of cells with Viobility™ Fixable Dye
- Viobility Fixable Dye was reconstituted in advance. The vial was warmed up to room temperature to avoid water condensation. One vial of lyophilized Viobility Fixable Dye was reconstituted by addition of 100 µL anhydrous DMSO to the dye vial and mixed until completely dissolved. The solution was aliquoted and stored at –20 °C under anhydrous (desiccant) conditions and protected from light for up to one month.
- Volumes given below were 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).
- Cell number was determined.
- Cells were washed by addition of 1 mL of 1× PBS and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of 1× PBS.
- 1 µL of Viobility Fixable Dye was added.
- Samples were mixed well and incubated 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. - Cells were washed by addition 1 mL PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- Step 6 was repeated.
- Surface staining was performed.
Surface staining
- Volumes given below were for up to 106 nucleated cells. When working with fewer than 106 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 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- A master mix with all antibody-fluorochrome conjugates except CD68 Antibody was prepared according to the dilution listed in the respective antibody data sheets.
▲Note: CD68 is an intracellular marker and will be detected after fixation and permeabilization of cells. - 100 µl antibody master mix was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
- Intracellular staining was performed.
Intracellular staining
- Reagents were prepared freshly. Failure to do so may lead to sub-optimal results.
- The required total buffer volumes were calculated beforehand; volumes will depend on the number of cells to be analyzed as well as the number of tests to be performed.
- Cells previously stained for surface markers were resuspended in 250 µL of Inside Fix solution from the Inside Stain Kit.
- Cells were mixed well and incubated for 20 minutes at room temperature.
- Cells were centrifuged for 5 minutes at 300×g.
- Supernatant was aspirated completely and the pellet was resuspended in 1 mL of PEB buffer.
- Cells were centrifuged for 5 minutes at 300×g and supernatant was aspirated completely.
- CD68 Antibody was diluted in Inside Perm according to the respective data sheet to a final volume of 100 µL and added to the cells.
- Cell suspension was incubated for 10 minutes at room temperature.
- 1 mL of Inside Perm buffer was added and cells were centrifuged for 5 minutes at 300xg.
- Supernatant was aspirated completely and the pellet was resuspended in 0.5-1 mL of PEB buffer.
- Flow cytometric analysis was performed using the MACSQuant® Analyzer.
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD11b | REA592 | Macrophages | VioBlue® | 450/50 (violet) |
| Viobility™ 405/520 Fixable Dye | - | Viability | - | 525/50 (violet) |
| F4/80 | REA126 | Red pulp macrophages | FITC | 525/50 (blue) |
| CD68 | REA835 | Red pulp macrophages | PE | 585/40 (blue) |
| CD3ε | REA606 | Cell type exclusion | PE-Vio® 615 | 655-730 (blue) |
| CD19 | REA749 | Cell type exclusion | PE-Vio® 615 | 655-730 (blue) |
| CD335 (NKp46) | REA815 | Cell type exclusion | PE-Vio® 615 | 655-730 (blue) |
| CD64 | REA286 | Red pulp macrophages | PE-Vio 770 | 750LP (blue) |
| CD45 | REA737 | Leukocytes | APC-Vio 770 | 750LP (red) |
| Mer | REA477 | Red pulp macrophages | APC | 655-730 (red) |
Additional reagents used
- Phosphate-buffered saline (PBS), pH 7.2
- PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
- Deionized or distilled water
- Inside Stain Kit (# 130-090-477)
- 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)
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130-112-856
CD68 Antibody, anti-mouse, PE, REAfinity™
REA835
ICFC, IF, IHC, MICS
30 µg in 200 µL (1)
USD 71.00
130-113-810
CD11b Antibody, anti-mouse, VioBlue®, REAfinity™
CD11b Antibody, anti-mouse, VioBlue
®
REA592
FC
30 µg in 200 µL (1)
USD 136.00
130-117-509
F4/80 Antibody, anti-mouse, FITC, REAfinity™
REA126
FC, MICS, IF, IHC
30 µg in 200 µL (1)
USD 54.00
