What is the Role of Pluribead in Monocyte Isolation For Dendritic Cell Maturation

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

DCs are highly motile immune cells that are widely distributed throughout tissues and represent a diverse group of cells with similar functions. Due to their high phagocytic activity and antigen processing capacity, they continuously sample the environment for antigens via endocytosis. In vivo, moDCs are only present when the body is inflamed, and they co-localize with any invading pathogen. This is in contrast to the normal steady-state presence of conventional DCs.

In both basic and applied research, such as the development of cancer immunotherapies, the generation and use of moDCs have been extensively described. For DC-associated in vitro studies, they are considered the gold standard. Traditionally, in vitro supplementation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL), -4 to generate immature DCs has aided monocyte differentiation into moDCs.

A key phenotypic difference between monocytes and moDCs is the persistent loss of CD14 expression (CD14low/-), which is usually accompanied by an increase in CD209 expression.

Human DC can be generated in vitro from CD14+ monocytes from peripheral blood, known as monocyte-derived DC (MoDC), or from CD34+ progenitors. Using blood dendritic cell antibodies, it has been demonstrated that DC can be isolated directly from peripheral blood (BDCA).

Although recent methodological advances in cell purification have provided a number of options for generating DC from various sources, each method should be thoroughly characterized before being used in clinical settings.Pluribeads can be used to isolate CD14+ cells using a positive selection method. This method is quick and yields a high number of pure cell populations.

About Pluribead

PluriBead is a one-of-a-kind cell enrichment technology that doesn’t depend on magnetic components. The procedure is straightforward: Your pluriBeads (with bound target cells) are sieved through a strainer; the pluriBeads with your target cells remain on top, while the unwanted cells pass through. You have your target cells ready after detaching.

Key features of Pluribead

No Sample Preparation: Use a sample volume of 200 l – 45 ml without gradient centrifugation, erythrolysis, or other procedures.
Use any sample material: whole blood, buffy coat, PBMC, secretion/excretion material, brain homogenate, spleen, liver, and so on.
Can be used on a Huge Range of Species: Isolate from humans, mice, rats, bovines, canines, sheep, and other species.
Fast Isolation: Cell enrichment can be started in five minutes
Gentle Isolation: A high yield of viable cells reduces the amount of sample required.
Universal pluriBeads: can be used with any external antibody.
Simultaneous Cell Isolation Using PluriBead Cascade: Separate two different cell types from the same sample material at the same time.
Sequential Cell separation: Isolate up to six different targets from a single sample using sequential cell isolation.

Read More: What Are The Different Types Of T Cells And The Unique Cell Separation Technologies?

Two Different Bead Sizes Available

S-pluriBead: For a small number of targets in a large sample volume (e.g. CTC).
M-pluriBead: Suitable for a large number of targets in a small amount of material (e.g. buffy coat)

PluriBead Principle

Mixing: pluriBead is added to the sample material.
Incubation: Target cells bind to pluriBead.
Separation: Unwanted cells are run through pluriStrainer – cell strainer Bound target cells to stay on top.
Detachment: Target cells are detached from the bead and run through a fresh sieve into a collecting tube.

About PluriPlix

PluriPlix is a non-electric universal mixer with no drive system that is designed to be used on top of a magnetic stirrer. Specifically designed for gentle cell mixing. Allows for two types of mixing: over-head and tilting-rotation (plus: adjustable rotation angle). It is intended for the incubation of fluid samples and has a maximum load capacity of 4 x 50 ml reaction tubes per sample holder.

The maximum weight per holder of 240 g should not be exceeded. A magnetic stirrer with a power output of 10 watts is recommended for maximum load operation. Because the driving energy is limited, overloading should be avoided. The user is not endangered by the input power.

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–

Non-magnetic with 280µl CD14 S-pluriBead anti-hu or 300µl M-pluriBead anti-hu

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. In the sample tube, add pluriBead CD14 for monocyte isolation and incubate for 20 minutes on a pluriPlix or wiping rolling mixer.

After cell isolation, resuspend the cells in 1ml of RPMI 1640-medium (+10 % FCS and 1x Pen/Strep) and determine the cell number. For the cultivation of monocytes in a 24-well cell culture plate, 1 million cells per well are used and incubated with 1ml monocytes-culture-medium (RPMI 1640 + 10 % FCS, 1x Pen/Strep, 2000 U/ml GM-CSF und 200 U/ml IL-4) at 37 °C and 5 % carbon dioxide.

Remove the medium after 24 hours and replace it with 1 mL fresh monocytes-culture-medium. The cells are then cultured for another four days. After 5 days, give the cells another 24 hours of stimulation with 100ng/ml LPS. The activated cells are then removed with trypsin, and the rate of monocyte maturation into dendritic cells is measured using CD1a, CD14, and CD83 fluorescent analysis.