Complete freund s adjuvant cfa

For CIA induction, Female DBA/1 mice were injected subcutaneously at the base of the tail with 100 µg of Chick type II collagen (Condrex, Seattle, WA, USA) emulsified in Freund’s complete adjuvant (CFA) (Chondrex, Seattle, WA, USA), followed 14 days later by a booster injection of the same Chick type II collagen (100 µg) emulsified in Freund’s incomplete adjuvant (Chondrex, Seattle, WA, USA), and mice injected with CFA/IFA only as a negative control. Which via the same route and following the protocol described by David et al (22 (link)). To assess the severity of arthritis, clinical symptoms were evaluated by means of a five-point scale: grade 0 = no swelling; grade 1 = paw with detectable swelling in a single digit; grade 2 = paw with swelling in more than one digit; grade 3 = paw with swelling of all digits and instep; and grade 4 = severe swelling of the paw and ankle.

The model was performed as previously described (26 (link)). CIA was induced in the mice at the age between 10 and 14 weeks. The immunization grade chick CII (2 mg/ml; #20012; Chondrex, Redmond, WA) was mixed gently with an equal volume of a 4 mg/ml Freund's complete adjuvant (CFA) (# 7001; Chondrex, Redmond, WA). Mice were anesthetized by isoflurane, and then intradermal injection was performed at multiple sites on the base of a tail (<50 μl/site). Experimental mice received 150 μl of CFA + CII emulsion, and control mice received only CFA or PBS. Three weeks after the first injection, mice received a booster injection of CFA + CII emulsion, CFA alone, or PBS. The mice were monitored and graded for arthritis severity (0 = normal; 1 = slight swelling and/or erythema; 2 = pronounced swelling; 3 = ankyloses) while performing the blind technique every other day.

1-Ethyl-3-(3-dimethylamino)propyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich Shanghai Trading Co, Ltd. (Shanghai, China). Poly(ethyleneglycol) (100) monostearate (S100) with PEG molecular weight 4400 g/mol was obtained from Sigma (Milwaukee, WI, USA). Methotrexate hydrate (MTX, purity 99%), doxorubicin (DOX, purity 98%), folate (FOL, purity ≥ 98%), catalase (from bovine liver, ≥3000 units/mg protein), hydrogen peroxide solution (30 wt % in H₂O), lipopolysaccharides (LPS), Trition X-100 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, purity > 99%) were all purchased from Shanghai Aladdin Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO), MTT, phosphate-buffered saline (PBS), 4′,6-diamidino-2-phenylindole (DAPI), folate-deficient Dulbecco’s modified eagle mediun (DMEM) medium (Hyclone^®), penicillin-streptomycin solution, trypsin-EDTA solution (Hyclone^®) and fetal bovine serum (FBS, Hyclone^®) were all provided by Sunshine Biotechnology Co., Ltd. (Nanjing, China). Chickens type II collage (2 mg/mL), complete Freund’s adjuvant (CFA, 4 mg/mL) and incomplete Freund’s adjuvant (IFA, 4 mg/mL) were purchased from Chondrex, Inc. (Redmond, WA, USA). All other chemicals and reagents were of HPLC grade.