BMDC Differentiation Cytokine Set, Mouse

1. Isolation of Murine Bone-Marrow Cells

1.1 Execute 6–8-week-old C57BL/6 mice (or strain specified in study plan) by cervical dislocation. Aseptically dissect both femora; remove adhering muscle with sterile scissors/forceps.

1.2 Disinfect bones in 75 % ethanol (2 min), then rinse twice with 10 mL ice-cold PBS.

1.3 Cut off epiphyses; flush marrow cavity with 5 mL cold PBS using a 25 G needle attached to a 10 mL syringe. Repeat until diaphysis appears white.

1.4 Pool marrow suspensions; filter through a 70 µm (200-mesh) nylon cell strainer into a 50 mL tube.

1.5 Centrifuge 1200 rpm (≈ 300 × g), 5 min, 4 °C; discard supernatant.

1.6 Resuspend pellet in 2 mL ACK red-cell lysis buffer; incubate 3–5 min at RT.

1.7 Add 10 mL PBS to stop lysis; centrifuge as above.

1.8 Wash once with 10 mL PBS; resuspend in complete RPMI-1640 + 10 % FBS (pen/strep 1 %, 2 mM L-Gln). Keep on ice.

2. In-vitro Differentiation of BMDC

2.1 Plate 1 × 10⁶ nucleated cells per mL in 6-well plates (3 mL/well) in complete medium supplemented with:

• GM-CSF -20 ng/mL

• IL-4- 20 ng/mL

This is designated Day 0 (D0).

2.2 Semi-media change every 48 h:

a. Remove 1.5 mL supernatant (non-adherent cells) into 15 mL tube.

b. Centrifuge 300 × g, 5 min; resuspend in 1.5 mL fresh complete medium containing fresh cytokines (same concentration).

c. Return cell suspension to original well.

2.3 On D6 harvest loosely adherent and firmly adherent cells:

• Pipette off supernatant; gently rinse wells with 2 mL PBS.

• Add 2 mL cold PBS, scrape with rubber policeman; pool all cells.

• Centrifuge 300 × g, 5 min; count.

• Re-plate at 1 × 10⁶ cells/mL in fresh complete medium + GM-CSF (20 ng/mL) + IL-4 (20 ng/mL).

These cells are “immature BMDC” (iBMDC).

2.4 Maturation (optional): On D8 add:

• TNF-α  20 ng/mL

• GM-CSF & IL-4 as above

Harvest on D10; these are “mature BMDC” (mBMDC).

3. Flow-Cytometric Validation of iBMDC (D6)

3.1 Harvest cells as in 2.3; wash once with PBS.

3.2 Count; adjust to 1 × 10⁷ cells/mL in FACS buffer (PBS + 2 % FBS).

3.3 Block Fc receptors: aliquot 100 µL/well (≈ 1 × 10⁶ cells) into 96-well V-bottom plate or FACS tube; add Mouse FcR Blocking Reagent (Mouse IgG); incubate 30 min on ice.

3.4 Stain surface markers (manufacturer’s titration):

Panel 1: PE anti-CD11c (clone N418, Cat. 117307) + APC anti-CD80 

Panel 2: PE anti-CD11c (as above) + APC anti-CD86 

Incubate 30 min, 4 °C, dark.

3.5 Wash twice with 200 µL FACS buffer; centrifuge 300 × g, 5 min.

3.6 Resuspend in 100 µL FACS buffer; add 7-AAD viability dye  5 µL per tube; incubate 5 min RT, dark.

3.7 Acquire on flow cytometer (≥ 20 000 events per sample).

Expected iBMDC phenotype: CD11c⁺ CD80^low CD86^low 7-AAD⁻.

4. Flow-Cytometric Validation of mBMDC (D10)

4.1–4.7 Identical procedure as Section 3, but use cells harvested after TNF-α maturation.

Expected mBMDC phenotype: CD11c⁺ CD80^high CD86^high 7-AAD⁻.
 

store in separate containers to reduce the number of freeze-thaw cycles.