This protocol describes preparation of a modified complete Freund's adjuvant (CFA) to be used for induction of adjuvant arthritis (AA) in rats. Commercial sources of CFA have not been found suitable for induction of AA. The particle size of the mycobacteria in the suspension may influence the success of disease induction; for this reason, large particles of mycobacteria must be broken up by grinding into smaller particles.
100 mg dried, heat-killed Mycobacterium tuberculosis (strain H37Ra) 10 ml incomplete Freund's adjuvant (IFA; Difco)
Pestle and ~7-cm-diameter roughened glass or porcelain mortar 15-ml plastic or glass tube
CAUTION: Wear gloves when handling the suspension of mycobacteria in oil, as it may be irritating and potentially sensitizing for humans.
1. Put 100 mg dried, heat-killed M. tuberculosis into a 7-cm-diameter roughened glass mortar.
2. Grind intensively >2 min with the pestle until color of dry mycobacteria changes from grey to white.
3. Add 3 ml IFA and continue grinding >1 min until mixture has changed into a paste.
4. Transfer paste to a 15-ml tube.
5. Add 3 ml IFA to mortar and resuspend remaining paste by grinding 30 sec. Transfer paste to the 15-ml tube.
6. Add remaining IFA and grind 30 sec. Transfer paste to the 15-ml tube. Store <1 month at 4°C.
Before using the suspension, it is important to carefully shake it to ensure even distribution of mycobacterial particles in different aliquots of adjuvant.
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