Macrophage Depletion Procedures and Literature Case Analysis

1. Introduction to Macrophage Depletion

Macrophages are the only type of cell found in every organ of the body, including the epidermis, cornea, and avascular joints. One of the important methods for studying their biological roles in vivo is macrophage depletion. By eliminating macrophages, researchers can gain a comprehensive understanding of their functions under pathological conditions.
Currently, the main approaches for macrophage depletion include generating macrophage-deficient animal models and using chemical agents such as clodronate liposomes. However, creating macrophage-deficient animal models is expensive and time-consuming. Clodronate liposomes (macrophage depletion agents) are currently the most mature, convenient, and cost-effective tool for effectively depleting macrophages in various tissues and organs such as the liver, spleen, lungs, and blood. This is the most widely used method for macrophage depletion to date.

2. Principle of Macrophage Depletion Agents

Clodronate is encapsulated into the aqueous phase of phospholipid bilayers to form clodronate liposomes. Clodronate cannot freely cross the phospholipid bilayer. When injected in vivo, clodronate liposomes are phagocytosed by macrophages. Within macrophage lysosomes, phosphatases degrade the liposomes, gradually releasing clodronate into the cell. Once clodronate accumulates to a certain concentration, irreversible damage occurs, triggering apoptosis. The clodronate released from the dead cells is excreted in the urine.

3. Recommended Administration Methods for Macrophage Depletion Agents

The optimal dosage may vary slightly depending on mouse strain. Dosage can be optimized based on the table below.

Table 1. Macrophage depletion administration methods in C57 mice

Administration	Details
Method	Intraperitoneal injection, intravenous injection, intranasal instillation, etc.
Dosage (C57 mice)	Single dose: 150–200 μL/mouse (20–25 g); Multiple doses: 150–200 μL/mouse (20–25 g) every 3–7 days. (Note: Dosage and interval vary by mouse strain and target tissue; values are not fixed. Adjust according to pilot experiments or relevant literature.)

4. Macrophage Markers

Macrophages exist in many subtypes, and their markers vary accordingly (see Table 2). In addition, macrophage markers differ between tissues and organs (see Table 3). Before conducting a macrophage depletion experiment, it is recommended to review the literature to identify the surface markers of macrophages in the target tissue and select the most appropriate marker for flow cytometric analysis.

Table 2. Markers of different macrophage subtypes

Reference: Klopfleisch R. Macrophage reaction against biomaterials in the mouse model - Phenotypes, functions and markers. Acta Biomater. 2016 Oct 1;43:3-13. doi: 10.1016/j.actbio.2016.07.003. Epub 2016 Jul 6. PMID: 27395828.

Table 3. Macrophage markers in different tissues

Macrophage Type	Tissue	Markers
Adipose tissue macrophages	Adipose tissue	CD45+, F4/80+, PPARγ+
Kupffer cells	Liver	B7-1/CD80^low/-, CD11b/Integrin αM^low, CD68/SR-D1+, F4/80^high, Galectin3/Mac-2+, PPARδ+, Siglec-1/CD169+
Alveolar macrophages	Alveoli	CD11b/Integrin αM^low/-, CD11c^high, CD68/SR-D1+, CD200R1+, DEC205/CD205^int, Dectin1+, F4/80^low, Galectin3/Mac-2+, MARCO+, MHC class II^low, MMR/CD206^high, PPARγ+, Siglec-F^high
Interstitial lung macrophages	Lung	CD11b/Integrin αM^int, CD11c-, CD68/SR-D1+, CD200R1^+/-, F4/80+, MHC class II^+/-, SiglecF-
Bone marrow macrophages	Bone marrow	CD169+, ER-HR3+, F4/80+, TRAP-, VCAM1/CD106+
Intestinal macrophages	Gastrointestinal tract	CD11b/Integrin αM+, CD11c^low/int, CX3CR1^high, F4/80+, FcγRI/CD64^+, MHC Class II^high

5. Literature Examples of Macrophage Depletion

Study 1

Mouse strain: C57BL/6J

Administration route: Intraperitoneal injection

Sampling site: Peripheral blood

Dosage: 200 μL/mouse

Antibody: F4/80

Method: A macrophage depletion model was established using clodronate liposomes, with 200 μL administered intraperitoneally per mouse. Twenty-four hours after injection, peripheral blood macrophages were quantified by flow cytometry.

Results:

Figure 1. Reduction of macrophages after intraperitoneal injection.

Reference: Xiong X, Chen S, Shen J, et al. Cannabis suppresses antitumor immunity by inhibiting JAK/STAT signaling in T cells through CNR2. Signal Transduct Target Ther. 2022;7(1):99. doi:10.1038/s41392-022-00918-y (IF: 18.187)

Study 2

Mouse strain: Female C57BL/6J

Administration route: Intraperitoneal injection

Sampling site: Peritoneal lavage fluid

Dosage: 15 mg/kg on days 0, 3, and 6

Antibody: CD11b, F4/80 double-positive

Method: Clodronate liposomes (15 mg/kg) or control empty liposomes (in 200 μL PBS) were injected intraperitoneally into 6-week-old female C57BL/6J mice on days 0, 3, and 6 to deplete macrophages. On day 3 or 6 after the last injection, 1×10^6 BMDMs in 100 μL PBS were injected intravenously to reconstitute macrophages. Flow cytometry was used to analyze macrophage depletion and reconstitution in peritoneal lavage fluid.

Results:

 

Figure 2. Flow cytometric analysis of macrophage depletion and reconstitution after liposome injection.

Reference: Zhang Z, Chen C, Yang F, et al. Itaconate is a lysosomal inducer that promotes antibacterial innate immunity. Mol Cell. 2022. doi:10.1016/j.molcel.2022.05.009 (IF: 17.970)

Study 3

Mouse strain: C57BL/6J

Administration route: Intravenous injection

Sampling site: Blood, mammary gland

Dosage: 200 μL/mouse

Antibody: F4/80

Method: Clodronate liposomes (0.15–0.2 mL/25 g) were administered intravenously at 200 μL per lactating mouse to deplete macrophages in blood and mammary glands. Flow cytometry was performed at 24, 48, and 72 h post-injection to quantify mammary gland macrophages.

Results

(a) Changes in monocyte proportion in blood detected by hematology analyzer.
(b) Changes in mammary gland macrophage proportion detected by flow cytometry.

Reference: Cai J, Peng J, Zang X, et al. Mammary Leukocyte-Assisted Nanoparticle Transport Enhances Targeted Milk Trace Mineral Delivery. Adv Sci (Weinh). 2022;9(26):e2200841. doi:10.1002/advs.202200841 (IF: 17.521)

Study 4

Mouse strain: C57BL/6

Administration route: Intraperitoneal injection

Sampling site: Peripheral blood

Dosage: 200 μL/mouse

Antibody: F4/80

Method: On day 3, clodronate liposomes (200 μL) were injected intraperitoneally to deplete macrophages. Flow cytometry was used to verify depletion.

Results:

Figure 4. Flow cytometric analysis of immune cell depletion.

Reference: Yu X, Long Y, Chen B, et al. PD-L1/TLR7 dual-targeting nanobody-drug conjugate mediates potent tumor regression via elevating tumor immunogenicity in a host-expressed PD-L1 bias-dependent way. J Immunother Cancer. 2022;10(10):e004590. doi:10.1136/jitc-2022-004590 (IF: 12.469)

6. Common Issues in Macrophage Depletion

Problem	Solution
How to set up control groups?	Use empty liposomes as a negative control.
Can the agent be used for in vitro depletion?	Yes, but it is more suitable for in vivo use. In vitro, released clodronate remains in the culture medium, whereas in vivo it has a short half-life and is cleared by the kidneys. Accumulated free clodronate in vitro may slowly enter cells.
Cause of rapid death after intravenous injection?	(1) Use at room temperature; (2) Mix well before use; (3) Avoid contamination or infection in animals.
How to determine injection site, dosage, timing, and frequency for different organs/tissues?	Base on specific experimental design; refer to literature or design your own scheme.
Why are ED1+ cells in spleen/liver not fully depleted after IV injection?	Mature macrophages in rats are ED1+/ED2+, while precursor cells without phagocytic function are ED1+/ED2−. Clodronate liposomes can completely deplete ED2+ cells, but only partially deplete ED1+ cells; depletion efficiency depends on the ratio of precursors to mature cells.
Reasons for suboptimal depletion?	(1) Verify clodronate concentration and absence of contaminants for each batch; (2) Store at 4–8 °C, avoid freezing or heating above 30 °C; (3) Use within 3 months of receipt.
Can IV injection volume be increased?	IV dose ≤0.1 mL/10 g body weight; intraperitoneal injection volumes can be slightly higher; subcutaneous injection volume depends on site capacity.

Related Products

Product Name	Specification	Cat No.
Clodronate Liposomes	5 mL/10 mL	40337ES08/10
Control Liposomes( PBS )	5 mL/10 mL	40338ES08/10