Info

Materials

Female or male BN rats 8 to 12 week old (e.g., Charles River Labs) HgCl2 (e.g., Sigma)

Distilled water, PBS, or 0.9% (w/v) NaCl, sterile Protein assay kit (Bio-Rad) or equivalent

Metabolic cages for housing rats 1-ml syringes with 26-G needles 50-ml conical polypropylene tubes, sterile

Additional reagents and equipment for rat identification (unit 1.5); anesthesia (unit 1.4); and euthanasia (unit 1.8); blood collection (unit 1.7); serum preparation (unit 2.4); determination of IgE (see Support Protocol 1) and autoantibody titers (see Support Protocols 2 and 3); and evaluation of arthritis symptoms (unit 15.4)

Prepare for HgCl2 treatment

1. Weigh the rats and identify them either by painting numbers on tail or punching ear

Test four or five rats per group (experimental and control).

2. Lightly anesthetize rats and collect ~500 pl blood from each animal by retroorbital puncture (units 1.7 & 2.4).

3. Allow blood to clot at 4°C, centrifuge samples 5 min at 800 x g, and collect serum (unit 2.4). Divide into 100-pl aliquots, then analyze serum to determine IgE (see Support Protocol 1) and autoantibody titers (see Support Protocols 2 and 3); alternatively, freeze serum samples at -20°C for later analysis.

This pretreatment serum sample serves as a negative control for serum determinations.

Sample may be stored up to several months at -20°C before analysis.

IgE is a highly sensitive parameter that increases after administration of as little as 5 jug

HgCl2 per 100 g body weight. Anti-laminin Ab production characterizes the autoimmune response but is not found at low doses of HgCl2.

4. House the rats individually in metabolic cages for 15 to 20 hr with free access to water but without food. Collect all urine produced by each rat during the 15 to 20 hr it is in the cage and record volume. Use 1 ml to determine protein content using a Bio-Rad protein assay (or equivalent) according to manufacturer's instructions; alternatively, freeze samples at -20°C for later analysis. Express the results as the amount of protein per 24 hr.

Note the urine volume and the date of collection.

Normally proteinuria is <20 mg/24 hr.

5. Working in a fume hood and wearing gloves, prepare a 1 mg/ml stock solution of HgCl2 by weighing 20 mg HgCl2 into a 50-ml conical polypropylene tube and dissolving it in 20 ml distilled water, PBS, or 0.9% NaCl.

The stock solution can be kept at room temperature. HgCl2 is light sensitive and will degrade; cover the tube with foil and discard after 1 week.

Mercuric Chloride-Induced Autoimmunity

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