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HumanKine®  G4 Dendritic Cell Generation Kit

Protocol:

HumanKine® G4 Dendritic Cell (DC) Generation Kit has been developed for the growth and generation of human dendritic cells (DCs). Using HumanKine G4 DC medium, human Dendritic Cells can be produced from peripheral blood mononuclear cells (PBMC) WITHOUT MEDIUM CHANGE and used either as immature DCs or further cultivated in to mature DCs. A certificate of analysis is included with the order.

Intended Use:

For research use only. Not intended for human or animal diagnostic or therapeutic uses.

Storage:

G4 DC Basal Medium: Store at 4ºC, protect from light.
G4 DC Cocktail (Lyophilized): Store at -80ºC.
Complete medium (G4 DC Cocktail added to G4 DC Basal Medium): Can be stored at 4ºC for up to 30 days in the dark. It is recommended to aliquot the complete medium into required working amounts and to avoid exposing the complete medium to 37ºC multiple times.

A. Complete Medium Preparation

G4 DC Basal Medium is composed of RPMI 1640 that has been supplemented with L-Glutamine, 25mM HEPES, 100 units Penicillin and 50µg/mL Streptomycin. G4 DC complete medium requires final concentration of 10% Fetal Calf Serum, 50µM beta-mercaptoethanol, and G4 DC Cocktail added to the G4 DC Basal Medium. Complete medium is stable for 30 days when stored at 4ºC in the dark.

1. For 500mL (100mL) complete medium, aseptically add 50mL (10mL) Fetal Calf Serum to G4 DC Basal Medium, HZ-9501-m (HZ-9101-m).
2. Aseptically add 5mL (1mL) of 5mM beta-mercaptoethanol to 500mL (100mL) medium.
3. Briefly centrifuge the G4 DC Cocktail vial prior to opening to draw contents to the bottom of the vial. Aseptically resuspend G4 DC Cocktail, HZ-9501-c (HZ-9101-c) in 0.5mL (0.1mL) sterile 1x PBS. Transfer the liquid cocktail to 500mL (100mL) of medium, rinse tube with complete medium and transfer liquid to 500mL (100mL) of medium. The final medium volume at this point should be approximately 555 mL (111 mL).

B. Monocyte Preparation

1. Isolate peripheral blood mononuclear cells from blood by Ficoll gradient centrifugation.
2. Isolate monocytes from peripheral blood mononuclear cells by using either Percoll gradient centrifugation (Monocyte purity ~90%) or using CD14 monocyte purification kit available from MiltenyiBiotec (Monocyte purity ~ 95–98%).

C. Immature Dendritic Cell Generation

This protocol is designed for use with product number HZ- 9501 (HZ-9101), HumanKine G4 Dendritic Cell Generation Kit. In this protocol, HumanKine GM-CSF and HumanKine IL-4 are used to generate human monocyte-derived DCs. This protocol DOES NOT REQUIRE MEDIUM CHANGE OR MEDIUM ADDITION over the 7 day incubation period.

1. Suspend monocytes in prewarmed (37ºC) G4 DC complete medium at 3-5x105cells/ml.
2. Culture the monocyte suspension in a humidified incubator with an atmosphere containing 5% CO2 for 7 days. DO NOT ADD ADDITIONAL MEDIUM OR CHANGE THE MEDIUM.
3. Harvest mildly adherent and floating cells by centrifuging at 400 x g for 5 minutes at room temperature. The resulting cells are monocyte-derived immature DCs that are highly phagocytic with the immature DC surface expression phenotype (CD14neg, CD83neg, CD80low, CD86low, MHC Iint, and MHCint) and can migrate in response to CCR5 agonist (RANTES), but not to CCR7 agonists (ELC/SLC).

D. Maturation of Immature Dendritic Cells

1. Aseptically, suspend immature DCs at 5x105cells/ml in fresh complete G4 DC medium.
2. Culture in the presence of LPS (0.1-1 µg/ml) or CD40 ligand (10-20 µg/ml) in a humidified incubator with an atmosphere containing 5% CO2 for 48 hours.
3. Mature DCs downregulate their phagocytic capacity, display surface marker phenotype typical of mature DCs (CD14neg, CD83pos, CD80int, CD86int, MHC Ihigh, and MHChigh) and migrate in response to CCR7 agonists (ELC/SLC), but not to CCR5 agonist (RANTES).
4. Alternatively, maturation can be induced by adding 100U/ml HumanKine TNF-alpha. (Cat. No. HZ-1014, typical ED50 0.05-5 ng/mL).

References::

1.  Romani et al.1994. J Exp Med 180: 83-93.
2. Yang D. et al. J Immunol. 2004. 173 : 6134-6142
3. Tedder TF and Jansen PJ. 1997. Current Protocol in Immunology. 7.32.1-7.32.6