BMDC (Bone Marrow-Derived Dendritic Cells) are dendritic cells derived from bone marrow. In mice, DCs primarily originate from the bone marrow, and hematopoietic precursor cells can be induced to differentiate into distinct DC subsets in vitro by adding specific combinations of cytokines to the culture medium. Dendritic cells (DCs) are the most potent professional antigen-presenting cells and serve as a critical bridge between innate and adaptive immunity. In vitro generation and manipulation of DCs represent a core technique in immunological research and immunotherapy development.

This cytokine kit includes recombinant mouse GM-CSF, recombinant mouse IL-4, and recombinant mouse TNF-α.

GM-CSF is the key factor that drives the proliferation and differentiation of bone marrow progenitor cells toward the myeloid DC lineage.

IL-4 primarily suppresses excessive macrophage outgrowth and promotes the acquisition of a more typical DC phenotype (e.g., high expression of MHC II and CD11c).

TNF-α provides a potent maturation signal that induces full maturation of BMDCs and is typically added during the late stage of differentiation.

Features

• High purity: >95% as determined by SDS-PAGE.
• Low endotoxin residue: < 1.0 EU per μg by the LAL method.
• High activity and high stability.

Specification

Components

The product set includes:

Storage

Store at –25 ~–15 °C. Stable for 1 year from the date of receipt.

After reconstitution, store at 2–8 °C under sterile conditions for up to 7 days.

After reconstitution, store at –85 ~–65 °C under sterile conditions for up to 3 months.

Application

Differentiation and maturation of mouse BMDCs for immunological research and immunotherapy.

Figures

1. BMDC Morphology (Day 9)

200×

       400 ×

Figure 1. Morphological characteristics of BMDCs on Day 9.

On Day 9 of differentiation, bone marrow–derived dendritic cells (BMDCs) displayed typical dendritic morphology, characterized by irregular cell bodies and multiple dendrite-like protrusions.

2. Flow Cytometry Analysis (Day 9)

Figure 2. Flow cytometric analysis of BMDC maturation on Day 9.

Flow cytometry analysis demonstrated high expression of dendritic cell maturation markers, including CD11c, MHC II, CD80, and CD86, confirming the successful generation of mature BMDCs.

Protocol for Induction and Differentiation of Mouse BMDCs

1. Isolation of Mouse Bone Marrow Cells

1.1 Euthanize a 6–10-week-old mouse by cervical dislocation. Aseptically remove all femurs and tibias, and thoroughly strip away surrounding muscle tissue using scissors and forceps.

1.2 Transfer the bones into a sterile biosafety cabinet. Place them in a sterile Petri dish containing 70% ethanol for 2–5 minutes for surface sterilization, followed by two washes with sterile PBS.

1.3 Transfer the bones to a new Petri dish containing fresh PBS. Cut off both ends of each bone. Using a syringe filled with PBS, insert the needle into the marrow cavity from both ends and flush out the bone marrow repeatedly until the bones become completely white.

1.4 Collect the bone marrow suspension and filter it through a 200-mesh nylon mesh.

1.5 Centrifuge the filtrate at 1200 rpm for 5 minutes and discard the supernatant.

1.6 Resuspend the cell pellet in 2 mL of 1× Ammonium-Chloride-Potassium (ACK) lysing buffer and incubate at room temperature for 3–5 minutes (maximum 10 minutes).

1.7 Add 10 mL of PBS to neutralize the lysing buffer, then centrifuge at 1200 rpm for 5 minutes and discard the supernatant.

1.8 Wash the cells once with PBS, then resuspend in RPMI-1640 complete medium supplemented with 10% FBS. The resulting suspension contains mouse bone marrow cells.

Preparation of ACK Red Blood Cell Lysing Buffer:

10× Stock Solution: Dissolve 82.9 g NH₄Cl, 10.0 g KHCO₃, and 0.37 g Na₂EDTA in 1 L distilled water. Filter-sterilize through a 0.22 μm membrane and store at 4°C for up to 6 months.

1× Working Solution: Dilute the 10× stock 1:9 with sterile distilled water immediately before use.

[Note]: ACK lysing buffer can be harmful to bone marrow cells; therefore, minimize the lysis time as much as possible.

2. Induction and Differentiation of BMDCs

2.1 Adjust the cell density to 1 × 10⁶ cells/mL in complete medium.

2.2 Plate 1 mL of cell suspension per well in a 24-well plate. Add recombinant mouse GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). Incubate at 37°C in a 5% CO₂ incubator. This is designated as Day 0.

2.3 Every 2 days, gently swirl the plate and replace half the culture volume with fresh medium supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL).

2.4 On Day 6, collect both non-adherent and loosely adherent cells. Centrifuge at 1200 rpm for 5 minutes and discard the supernatant.

2.5 Resuspend the cell pellet in RPMI-1640 complete medium (with 10% FBS), count the cells, and adjust the concentration to 1 × 10⁶ cells/mL. Add GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). These are immature BMDCs.

2.6 Plate the cells in 100 mm dishes (up to 10 mL per dish) or 6-well plates (2 mL per well).

2.7 Continue culture at 37°C in 5% CO₂ for 1–2 additional days.

3. Full Maturation of BMDCs

3.1 On Days 7–8, harvest the immature BMDCs and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant.

3.2 Resuspend the cell pellet in RPMI-1640 complete medium, count the cells, and adjust the concentration to 1 × 10⁶ cells/mL. Add recombinant mouse TNF-α (20 ng/mL), GM-CSF (20 ng/mL), and IL-4 (20 ng/mL).

3.3 Incubate at 37°C in 5% CO₂ for 2 days.

3.4 On Days 9–10, collect both suspended and adherent cells. These are fully mature dendritic cells.

Documents:

Safety Data Sheet

92641_MSDS_HB260122_EN.PDF

Manuals

92641_Manual_Ver.EN20260122.pdf