Complete Freund's adjuvant (CFA) was prepared by re-suspending 4 mg of heat-denatured mycobacterium (Chondrex, LLC, Seattle, WA) in 1 mL of incomplete Freund's adjuvant (BD Biosciences). Bovine type II collagen (CII, 2 mg/mL) was emulsified with CFA at a ratio of 1:1 and then injected intradermally into the tail (1.5 cm from the base) of DBA1/J mice to induce collagen-induced arthritis. To determine the intervention effects, mice were divided into different groups randomly, with or without a single intravenous injection of 2 × 10^6 GMSC or 2 × 10^6 CD4 + T cells that had been activated in vitro with anti-CD3/CD28 antibodies on day 14 after immunization. Alternatively, a similar dose of human dermal fibroblast cell line (ATCC, #PCS-201-012) or dermal fibroblasts were injected intravenously as a control of GMSC.
Heat denatured mycobacterium
Manufactured by Chondrex
Sourced in United States
Heat-denatured mycobacterium is a laboratory-produced material that has been subjected to high temperatures to denature the proteins within the mycobacterium cells. This process alters the structure and function of the mycobacterium, rendering it unable to replicate or cause infection. The material is commonly used in research applications as a non-viable alternative to live mycobacterium samples.
Automatically generated - may contain errors
6 protocols using heat denatured mycobacterium
1
Collagen-Induced Arthritis Intervention
2
Murine Model of Collagen-Induced Arthritis
3
Rat Model of Collagen-Induced Arthritis
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Chicken type II collagen (Chondrex, Redmond, WA, USA) was mixed with Freund's complete adjuvant containing 4 mg/mL heat-denatured mycobacterium (Chondrex) to prepare an emulsion. On Day 0, the emulsion was injected intracutaneously into the toes and tail roots of rats to establish the CIA rat model. On Day 7, the rats were challenged in the same manner. After presenting secondary joint swelling on Day 14, the CIA rats were randomly divided into vehicle-treated CIA group (CIA-Veh), 2.5 mg/kg/day of indomethacin (Linfen Qilin Pharmaceutical Co., Ltd., Linfen, China) treated group (CIA-Indo), 15 mg/kg/day of Paroxetine (China-US Tianjin Shike Pharmaceutical, Tianjin, China) treated group (CIA-PAR) and 0.5 mg/kg/3 day of methotrexate (MTX) (Tonghua Maoxiang Pharmaceutical, Tonghua, China) treated group (CIA-MTX) and treated for 14 days. Body weight change, global assessment, arthritis index, swollen joints count, and secondary paw swelling were assessed every three days by two blinded, independent observers.
4
Collagen-Induced Arthritis Model in Mice
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For CIA induction, bovine type II collagen (CII; Chondrex) was emulsified with complete Freund's adjuvant (CFA) containing 4 mg/ml heat‐denatured mycobacterium (Chondrex, LLC) at a ratio of 1:1 (100 µg/mouse) and then injected intradermally into the tail (1.5 cm from the base) of DBA1/J mice to induce CIA (Day 0), followed by a booster immunization with 100 µg CII in incomplete adjuvant (IFA; Chondrex) at Day 21.36 At the moment of the boost (Day 21), GMSC (1 × 106/mouse) and GMSC‐Exo (165 µg/mouse) were injected into CIA mice (n = 6) via the lateral tail vein. In the control group, mice received equal PBS injection (n = 6).
Mice were monitored twice weekly for signs of arthritis based on arthritis scores and paw swelling. Each paw was evaluated and scored individually using a 0 to 4 scoring system: 0 = no damage; 1 = paw with detectable swelling in a single digit; 2 = paw with swelling in more than one digit; 3 = paw with swelling of all digits and instep; and 4 = severe swelling of the paw and ankle.15 On Day 56, all mice were euthanized, and peripheral blood, spleen, axillary and mesenteric lymph nodes (LN), and limbs were collected for further studies.
Mice were monitored twice weekly for signs of arthritis based on arthritis scores and paw swelling. Each paw was evaluated and scored individually using a 0 to 4 scoring system: 0 = no damage; 1 = paw with detectable swelling in a single digit; 2 = paw with swelling in more than one digit; 3 = paw with swelling of all digits and instep; and 4 = severe swelling of the paw and ankle.
5
Rat Model of Collagen-Induced Arthritis
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Chicken type II collagen (Chondrex, Redmond, WA, USA) was mixed with Freund's complete adjuvant containing 4 mg/mL heat-denatured mycobacterium (Chondrex) to prepare an emulsion. On Day 0, the emulsion was injected intracutaneously into the toes and tail roots of rats to establish the CIA rat model. On Day 7, the rats were challenged in the same manner. After presenting secondary joint swelling on Day 14, the CIA rats were randomly divided into vehicle-treated CIA group (CIA-Veh), 2.5 mg/kg/day of indomethacin (Linfen Qilin Pharmaceutical Co., Ltd., Linfen, China) treated group (CIA-Indo), 15 mg/kg/day of Paroxetine (China-US Tianjin Shike Pharmaceutical, Tianjin, China) treated group (CIA-PAR) and 0.5 mg/kg/3 day of methotrexate (MTX) (Tonghua Maoxiang Pharmaceutical, Tonghua, China) treated group (CIA-MTX) and treated for 14 days. Body weight change, global assessment, arthritis index, swollen joints count, and secondary paw swelling were assessed every three days by two blinded, independent observers.
6
Collagen-Induced Arthritis Model in Mice
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For CIA induction, bovine type II collagen (CII; Chondrex, Redmond, WA) was emulsi ed with complete Freund's adjuvant (CFA) containing 4 mg/ml heat-denatured mycobacterium (Chondrex, LLC, Seattle, WA) at a ratio of 1:1 (100 ug/mouse) and then injected intradermally into the tail (1.5 cm from the base) of DBA1/J mice to induce CIA, followed by a booster immunization with 100 ug CII in incomplete adjuvant (IFA, Chondrex, Redmond, WA) [33] . At the moment of the boost (day 21), GMSC (1×10 6 /mouse) and GMSC-Exo (165 ug/mouse) were injected into CIA mice (n = 6) via the lateral tail vein. In the control group, mice received equal PBS injection (n = 6).
Mice were monitored twice weekly for signs of arthritis based on arthritis scores and paw swelling. Each paw was evaluated and scored individually using a 0 to 4 scoring system: 0 = no damage; 1 = paw with detectable swelling in a single digit; 2 = paw with swelling in more than one digit; 3 = paw with swelling of all digits and instep; and 4 = severe swelling of the paw and ankle [34] . At the day 56, all mice were euthanized and peripheral blood, spleen, lymph nodes (LN), and limbs were collected for further studies.
Mice were monitored twice weekly for signs of arthritis based on arthritis scores and paw swelling. Each paw was evaluated and scored individually using a 0 to 4 scoring system: 0 = no damage; 1 = paw with detectable swelling in a single digit; 2 = paw with swelling in more than one digit; 3 = paw with swelling of all digits and instep; and 4 = severe swelling of the paw and ankle [34] . At the day 56, all mice were euthanized and peripheral blood, spleen, lymph nodes (LN), and limbs were collected for further studies.
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