By Andras Perl
This choice of with no trouble reproducible molecular strategies and similar in vitro/in vivo version platforms can be utilized to discover the factors of autoimmunity, in addition to how most sensible it can be regulated. there are ways to evaluate immunological and biochemical pathways appropriate for pathogenesis and to set up and verify numerous autoimmune ailments, together with arthritis, lupus, diabetes, a number of sclerosis, myocarditis, thyroiditis, scleroderma, uveitis, and vitiligo.
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Additional info for Autoimmunity: Methods and Protocols (Methods in Molecular Medicine)
2 –2 mL) with prewarmed, filtered RPMI-1640 containing 1% FBS. 5 µg/ mL) or the isotype control mIgG2a, followed by 10 µg/mL of goat antimouse antibody at 130 s. The intracellular calcium response was continuously monitored for 10 min with intermittent shaking. 2. BIO-RAD PROTEIN ASSAY 1. Take a flat-bottom, 96-well culture plate. 2. Pipet a row of 0, 2, 4, 6, 8, and 10 µL protein standard (1 mg/mL BSA in distilled water) and add 10, 8, 6, 4, 2, and 0 µL, respectively, of distilled water to make up the final volume to 10 µL.
Incubate the cells at 37°C for 10 min in a water bath. 3. Stimulate the cells with the addition of 1 µg/mL OKT3 for 0, 1, or 2 min at 37°C. (Antibody 2Ad2a2:IgM 1:200 dilution or UCHT1 10 µg/mL antibody can be substituted for OKT3. ) 4. 5 mL ice-cold 2X stop buffer (50 mM TrisHCl, 100 mM NaCl, 100 mM NaF, 2 mM Na3VO4, 10 mM EDTA, 10 mM sodium pyrophosphate, 2 mM phenylmethane sulfonyl fluoride (PMSF), 20 µg/mL leupeptin, and 20 µg/mL aprotinin). 5. 2M PMSF; 5 µL 200 mM sodium vanadate) containing 1% Nonidet P-40 (Sigma Aldrich).
1% Tween-20). 5. Wash the membrane in PBS or TBS once. 6. DETECTION 1. Warm and mix enhanced chemiluminescent (ECL) reagents 1 and 2 (Amersham Pharmacia) and mix mL of each to make 8 mL reagent per blot. 2. Incubate the membrane in the ECL reagent for 1 min. Make sure that the reagent spreads over the entire membrane. 3. Drain the ECL reagent from the membrane, dry the membrane gently, and then put it between a plastic envelope or saran wrap and place it on a detection cassette. 4. Cut Kodak X-ray film (Sigma Aldrich) to the appropriate size, bend the upper right-hand corner to identify the position, put the film on the membrane, expose for 10 s, and expose a second film for 1 min.
Autoimmunity: Methods and Protocols (Methods in Molecular Medicine) by Andras Perl