Dendritic cells are typically isolated in vitro from CD14+ monocytes (MoDCs). Let's look at how pluribead helped with monocyte separation for dendritic cell maturation.

Monocyte-derived dendritic cells (moDCs) are a subset of Dendritic Cells (DCs) that are commonly used in immunological studies following the mononuclear cell separation directly from circulation. Human moDCs can be generated in vitro from peripheral blood CD14+ monocytes or CD34+ progenitors.

Pluribead
A unique cell enrichment method that doesn't rely on magnetic components is called PluriBead. The process is easy to follow: Your pluriBeads are sieved through a strainer with your bound target cells on top, while the unwanted cells fall through to the bottom. You have your target cells ready after detaching.

Two Different Bead Sizes Available

S-pluriBead: It is used for a small number of targets in a large sample volume.

M-pluriBead: A versatile material that can be used for many targets while using less material (e.g. buffy coat)

Monocyte isolation, differentiation, and stimulation protocol

PluriSelect products required:

CD14 S-pluriBead anti-hu
CD14 M-pluriBead anti-hu
S-pluriBead Mini Reagent Kit
M-pluriBead Mini Reagent Kit
pluriPlix 3-in-1 Mixer

Sample volume- 10 ml human buffy coat

Isolation method-
With 280l CD14 S-pluriBead anti-HU or 300l M-pluriBead anti-HU, non-magnetic

Yield-
~6 x 106 monocytes with S-pluriBead
~12 x 106 monocytes with M-pluriBead

Vitality->92% (trypan blue staining)

Purity-~97%

The Process
Wash sample material twice with provided Washing Buffer (PBS + 0,05 % BSA and 2 mM EDTA, pH 7,4) in order to reduce soluble CD14. The sample tube should have PluriBead CD14 for monocyte isolation added before being incubated for 20 minutes on a pluriPlix or wiping rolling mixer.

Determine the cell number after resuspending the separated cells in 1 ml of RPMI 1640-medium (+10% FCS and 1x Pen/Strep). In a 24-well cell culture plate, monocytes are grown with 1 million cells per well in 1 ml of monocyte culture medium (RPMI 1640 + 10% FCS, 1x Pen/Strep, 2000 U/ml GM-CSF, and 200 U/ml IL-4) at 37 °C and 5% Carbon dioxide.

After 24 hours, discard the medium and add 1 mL of brand-new monocytes-culture-medium in its place. After that, the cells are cultured for a further four days. After five days, stimulate the cells for another 24 hours with 100ng/ml LPS. The rate of monocyte maturation into dendritic cells is then assessed using CD1a, CD14, and CD83 fluorescent analysis after the activated cells have been removed with trypsin.

This emphasizes the significance of selecting a monocyte isolation method appropriate for the subsequent experiments.

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