This article describes a method to induce systemic inflammation in mice using a mycobacterial emulsion. The process involves subcutaneous injection of the emulsion, which triggers an immune response leading to inflammation.
To induce systemic inflammation — a widespread inflammatory response, begin by positioning an anesthetized mouse with its dorsal side up. Take a syringe containing mycobacterial emulsion of mycobacterial antigen-adjuvant depots — a localized oil reservoir of the antigen-adjuvant mixture.
Pierce the mouse's epidermis and dermis skin layers and inject the antigen-adjuvant emulsion subcutaneously proximal to the inguinal lymph node. Post-injection, the depot continuously releases antigens into the surrounding tissue — providing prolonged exposure of the antigens to the immune system.
Antigen-presenting cells, or APCs, in the tissue, interact with injected mycobacterial antigens, internalize, and process them into peptides. These peptides are then presented on the surface of APCs using major histocompatibility complexes, triggering the release of pro-inflammatory cytokines and chemokines.
This process generates localized inflammation and triggers the opening of lymphatic vessels, enabling the entry of immune cells including antigen-carrying APCs into the lymphatic system and reaching the inguinal lymph node.
In lymph nodes, T cell receptors of helper T cells interact with antigens on the APCs' surface and become activated. The activated T cells release pro-inflammatory cytokines, which travel through lymphatic vessels and get released into various other tissues.
The released pro-inflammatory cytokines attract the immune cells to these tissues, inducing systemic inflammation.
To begin, unclamp the body of the sonicator converter unit and clean the probe with a 70% alcohol swab. Then, switch the sonicator on, and adjust the power setting to 4, by turning the power control knob.
Next, to generate a fine suspension of the Mycobacterium tuberculosis H37Ra in PBS, immerse the probe's tip into the PBS-containing mycobacterial powder. Sonicate the mixture on ice for 30 seconds, then, pause for 30 seconds, and repeat for a total of 5 minutes to fully disperse the powder into an even suspension without heating the liquid. Next, add 2.5 milliliters of Freund's incomplete adjuvant to the mixture, and repeat the sonication process on ice until the emulsion forms a toothpaste-like consistency.
For the subcutaneous injection, load 200 to 300 microliters of the mycobacterial emulsion in a 1-milliliter syringe. Expel the air from the syringe, and continue filling the syringe, with intermittent inverting and tapping, until filled.
Next, place the subcutaneous injections on either the dorsal surface of the hips, or on the ventral surface of the legs proximal to the region of the inguinal lymph nodes of a 6 to 10-week-old, anesthetized, C57Black/6J mouse. Carefully insert the needle, taking care not to penetrate the muscle, and inject 50 microliters of the emulsion into the subcutaneous space. Do not remove the needle immediately to allow the thick emulsion to be fully injected.
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