Immunophenotyping of dendritic cells from human whole blood using flow cytometry
Miltenyi Biotec-tested panel 10 (MBTP 10)
Miltenyi Biotec-tested panel 10 (MBTP 10)
A whole blood sample obtained from a healthy donor was analyzed for dendritic cell subsets.
Protocol
This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.
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).
- Erythrocytes were lysed using Red Blood Cell Lysis Solution.
- Cells were washed and cell number was determined.
- Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- A master mix with all antibodies was prepared according to the dilutions mentioned in the respective antibody data sheets.
- 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.
- Cells were resuspended in 100 µL of PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.
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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.
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD45 | REA747 | Leukocytes | VioGreen™ | 525/50 (violet) |
| CD303 (BDCA-2) | REA693 | Plasmacytoid dendritic cells | PE | 585/40 (blue) |
| CD14 | REA599 | Cell type exclusion | FITC | 525/50 (blue) |
| CD20 | REA780 | Cell type exclusion | FITC | 525/50 (blue) |
| CD123 | REA918 | Plasmacytoid dendritic cells | APC-Vio® 770 | 750LP (red) |
| CD141 (BDCA-3) | REA674 | Conventional dendritic cells 1 | APC | 655-730 (red) |
| CD1c (BDCA-1) | REA694 | Conventional dendritic cells 2 | PE-Vio 770 | 750LP (blue) |
| FCεR1α | CRA1 | Conventional dendritic cells 2 | VioBlue® | 450/50 (violet) |
| 7-AAD Staining Solution | - | Viability | - | 655-730 (blue) |
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.
- Red Blood Cell Lysis Solution (# 130-094-183)
- 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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REA694
FC
EUR 356,00
REA693
FC
EUR 190,00
REA674
FC, MICS, IF, IHC
EUR 329,00
REA918
FC
EUR 356,00
