Macrophages are found in almost every organ — from the epidermis and cornea to the liver, spleen, and even avascular joints. As central regulators of immunity and homeostasis, these cells are indispensable for understanding both normal physiology and disease.
Clodronate liposomes are the most established, convenient, and cost-effective tool for depleting macrophages in vivo, offering a robust approach for immunology and pathophysiology research.
This FAQ guide summarizes key principles, practical tips, and common troubleshooting strategies to help you design and execute macrophage depletion studies with confidence.
I. Mechanism Overview — How Clodronate Liposomes Work
- Principle: Clodronate liposomes are selectively engulfed by phagocytic cells (macrophages, dendritic cells). Once internalized, the liposome membrane is degraded, releasing clodronate into the cytoplasm.
- Effect: Intracellular clodronate triggers macrophage apoptosis, effectively depleting phagocytic cells while sparing non-phagocytic populations.
- Selectivity: Tissue distribution and depletion efficiency vary depending on injection route (i.v., i.p., intranasal, intracerebral, etc.).
II. Experimental Setup — Dosing and Administration
|
Target Organ / Macrophage Type |
Recommended Administration (20–25 g mouse) |
Notes / Tips |
|
Spleen (Red Pulp Macrophages) |
• Single dose: 200 µL/mouse (i.v. or i.p.)• Long-term: 150 µL initially, 100 µL every 2 weeks |
Mix thoroughly before use. Maintain dosing interval for consistent depletion. |
|
Liver (Kupffer Cells) |
200 µL (i.v. or i.p.) per mouse; long-term as above |
Suitable for hepatic inflammation or infection models. |
|
Lung (Alveolar Macrophages) |
Combine i.v. (150–200 µL) with intratracheal/nasal (50 µL) routes |
Dual delivery enhances depletion in airways and alveoli. |
|
Lymph Nodes |
100–200 µL/mouse (i.v. or i.p.) |
Refer to literature for optimal timing and marker selection. |
|
Brain (Microglia) |
10 µL/mouse or 50 µL/rat via intracerebroventricular injection |
Use stereotaxic guidance for accuracy. |
|
Blood (Monocytes) |
150–200 µL/mouse (i.v.); max depletion at 24 h |
Monitor at 24 h post-dose; flow cytometry recommended. |
III. Operation FAQ — Handling and Best Practices
Q1. How should Clodronate Liposomes be stored?
Store at 4°C. Do not freeze or dilute. Gently invert before use; avoid vortexing to maintain liposome integrity.
Q2. Can I prepare aliquots for later use?
Avoid multiple freeze–thaw cycles. If aliquots are needed, keep them at 4°C and use within one month.
Q3. What markers are suitable for validation?
- F4/80: blood, spleen, liver
- CD68 / CD11b: lung, lymph nodes
- Iba1: brain (microglia)
Q4. How long does depletion last?
Typically 2–4 days in circulation, 5–7 days in tissues. Recovery time depends on the organ and strain.
Q5. Can I increase the injection volume for better depletion?
For i.v. injection, do not exceed 0.1 mL per 10 g body weight. Slightly larger volumes are acceptable for i.p. injection.
IV. Troubleshooting — When Things Don’t Go as Planned
Q1. The mice died after injection — why?
- Suspension not homogeneous (mix gently before injection)
- Infection risk increases after macrophage clearance
- Maintain aseptic conditions during injection
Tip: Some studies recommend ampicillin (1 mg/mL) or doxycycline (200 mg/kg feed) to reduce infection risk.
Q2. No depletion detected in flow cytometry.
- Sampling time too late (depletion peaks at 24 h)
- Inappropriate marker (check tissue-specific ones)
- Liposomes not fully mixed
Q3. Blood macrophages recovered too fast.
That’s normal — monocytes in circulation typically recover within 3 days. For prolonged depletion, consider repeated dosing.
Q4. Can Clodronate Liposomes affect other immune cells?
They primarily target macrophages, but phagocytic dendritic cells can also be transiently affected. Always confirm cell populations via flow cytometry.
V. Literature Reference & Further Reading
- van Rooijen N, Sanders A. Elimination, blocking, and activation of macrophages: three of a kind? J Immunol Methods. 1994.
- Van Rooijen N, Hendrikx E. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods. 2010.
- Choi J et al. Selective depletion of macrophages using clodronate liposomes in vivo. Methods Mol Biol. 2017.
Clodronate Liposomes remain the gold standard for macrophage depletion in vivo — combining efficiency, selectivity, and ease of use. By understanding the mechanism, choosing the right route, and following good experimental practices, researchers can achieve consistent, reproducible results in immune and disease models.
Yeasen’s Clodronate Liposomes offer high batch-to-batch consistency and proven performance across mouse, rat, and zebrafish models — empowering your next macrophage-targeting study.
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