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Complete freund s adjuvant cfa

Manufactured by InvivoGen
Sourced in France

Complete Freund's adjuvant (CFA) is a laboratory reagent used to enhance the immune response in experimental animals. It is composed of mineral oil, emulsifiers, and heat-killed Mycobacterium tuberculosis. CFA functions as an immunostimulant, triggering a strong cellular and humoral immune response when administered along with an antigen.

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4 protocols using complete freund s adjuvant cfa

1

Anti-mullet Immunoglobulin Production in Mice

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The production of anti-mullet immunoglobulin in mice was performed according to Sharon et al. [31 (link)]. In short, 200 µL blood was withdrawn from the caudal vein of three anesthetized sick mullets (presenting higher levels of antibodies) using 1 mL syringes with a 30× g needle. Blood samples were incubated overnight at 4 °C and then centrifuged at 3500× g at 4 °C for 30 min; 100 µL of mullet serum was transferred to a new tube and the rest was frozen at −80 °C. The 100 µL of mullet serum was incubated with 40 µL of protein A/G covalently immobilized on Sepharose beads (ABCam, Cambridge, UK) and shaken for 2 h at room temperature (RT). The protein A/G beads were washed three times in PBS and centrifuged at 14,000× g for 1 min at RT; the supernatant was discarded, and the pellet with the proteins was resuspended in PBS at a final volume of 200 µL and emulsified (1:1, v/v ratio) in complete Freund’s adjuvant (CFA, Invivo Gen, Toulouse, France).
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2

OVA-Induced Allergic Response in Mice

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C57BL6 mice were immunized against OVA (50 μg – Grade IV, Sigma Aldrich) in Complete Freund's Adjuvant (CFA – InvivoGen). After 7 days, MutuDCs were pulsed with 100 μg/mL of OVA overnight, washed with PBS twice, and 3 × 106 cells were transferred to immunized mice by intraperitoneal (i.p.) injection. When IL10-DCs and IL35-DCs were transferred in combination, they were mixed only a few minutes before the injection, at 1:1 ratio. One week later, mice were challenged with 25 μL of heat-aggregated OVA (20 mg/mL – 500 μg/animal) in one footpad and the same volume of PBS was injected in the contralateral footpad as a control. Footpad thickness was measured with a dial thickness gauge (Mitutoyo) multiple times for 72 h. Blood, lymph nodes and spleen were collected and processed as mentioned above. Total cells were re-stimulated ex vivo with 100 μg/mL OVA for 24 h.
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3

Murine and Human Cell Culture

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Isolated murine and human cells were cultured with sterile RPMI 1640 supplemented with L-Glutamine, Penicillin, Streptomycin, 25mM Hepes (Lonza, Walkersville, MD).
Nickel (II) chloride (NiCl2), cobalt (II) chloride (CoCl2), phorbol 12-myristate 13-acetate (PMA), ionomycin, phytohemagglutinin (PHA), and Mitomycin-C were purchased from Sigma Aldrich (St. Louis, MO). NiCl2 and CoCl2 were reconstituted in sterile water and stock solutions were freshly prepared for each in vitro and in vivo experiment. Complete freund’s adjuvant (CFA) was purchased from InvivoGen (San Diego, CA).
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4

LCMV Infection and Immunization Protocol

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Mice were infected by intraperitoneal injection with 2×105 PFU of LCMV Armstrong or immunized by intramuscular (quadriceps) injection with 2–10 μg recombinant LCMV glycoprotein with addition of Addavax (InvivoGen) adjuvant at a 1:1 ratio, with 50 μg VacciGrade Poly I:C (HMW) (InvivoGen) adjuvant, with 50 μg VacciGrade CpG ODN 1826 (InvivoGen) adjuvant, with Aluminum hydroxide gel (Alhydrogel®, also known as alum) adjuvant 2% (InvivoGen) at a 1:1 ratio, or Complete Freund’s Adjuvant (CFA, 1 mg/ml of heat-killed dried Mycobacterium tuberculosis strain H37Ra, InvivoGen) at a 1:1 ratio. Recombinant LCMV Glycoprotein (GP)-expressing 293A cells were kindly provided by Dr. Carl Davis (Emory University), and recombinant GP was purified from supernatants as described previously (44 (link)).
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