Hello

I think this media is abetter to cell culture.

1. What is the best culture medium and activators for culturing plasma cells and plasmablasts from mouse spleen for 1-2 weeks?

To maintain plasma cells and plasmablasts from mouse spleen for an extended period, an appropriate culture medium and specific activators are required.

Recommended conditions:

Culture medium:

RPMI-1640 supplemented with 10-15% FBS (Fetal Bovine Serum) + 1% Penicillin/Streptomycin

Addition of β-Mercaptoethanol (50 µM) to maintain cell viability

Glutamine (2 mM) to support growth

Essential activators:

IL-21 and IL-4 to promote plasmablast proliferation

LPS (Lipopolysaccharide) 10 µg/ml to induce B cell differentiation into plasma cells

CD40L (50 ng/ml) to enhance cell survival

Environmental conditions:

Temperature: 37°C

CO₂ level: 5%

Medium change: every 2-3 days

Thank you, Marwa Saabneh! I really appreciate your response. Could you please share the reference for this information?

To successfully isolate and culture plasma cells (PCs) and plasmablasts (PBs) from primary mouse spleen B-cells, you will need a specialized culture system that supports their survival and function. Here’s a detailed plan:

1. Media Selection

Plasma cells and plasmablasts are terminally differentiated B cells that require specific growth factors and survival signals. A customized media rich in essential cytokines is required.

Recommended Media:

• Base Media:RPMI 1640 (with HEPES for buffering) 10% FBS (or 20% if viability is poor) 1X Penicillin/Streptomycin 2 mM L-glutamine 50 μM 2-mercaptoethanol (BME) (for reducing oxidative stress)
• Key Cytokines and Supplements:IL-6 (10–50 ng/mL) – Essential for plasma cell survival. IL-21 (10–50 ng/mL) – Enhances plasma cell differentiation and survival. BAFF (10–50 ng/mL) – Provides additional B-cell survival signals. APRIL (5–10 ng/mL) – Supports long-term plasma cell maintenance. Insulin-Transferrin-Selenium (ITS) – Supports long-term cell health.

Optional:

• SCF (Stem Cell Factor, ~10 ng/mL) – May support survival in some conditions.
• IFN-γ (optional, ~10 ng/mL) – May enhance IgG secretion.

2. Activation Before Culture (If Needed)

• If you are differentiating B cells into plasmablasts, pre-activation may be required using:LPS (5–10 μg/mL) α-CD40 (1–10 μg/mL) + IL-4 (10–20 ng/mL) CpG (1–3 μg/mL) Anti-IgM (10 μg/mL) or BCR ligands
• If you are directly isolating CD138+ plasma cells, no activation is needed; you can move directly to culture in plasma cell media.

3. Cell Isolation & Immediate Culture

• Step 1: Isolate Total B CellsUse magnetic sorting (MACS) or FACS to isolate total B220+ B cells from spleen.
• Step 2: Enrich for CD138+ Plasma Cells & PlasmablastsUse CD138 microbeads (MACS) or FACS sorting (CD138+ B220low/-).
• Step 3: Immediate Culture in Plasma Cell-Specific MediaPlasma cells can be cultured immediately after isolation. Plasmablasts may require additional activation signals (e.g., IL-21, CD40L).

4. Culture Conditions

• Plating Density: ~0.5–1 x 10⁶ cells/mL
• Incubation: 37°C, 5% CO₂
• Media Changes: Every 2–3 days (replace 50% of media to maintain cytokine levels).
• Viability Monitoring: Use Trypan Blue or Annexin V/PI staining.

Expected Outcomes

• Plasmablasts will proliferate but may not survive long-term.
• Plasma cells can survive 1–2 weeks with proper cytokine support.
• Adding stromal feeder cells (e.g., OP9 or CD40L-expressing fibroblasts) can enhance survival.