By Andras Perl
This number of simply reproducible molecular concepts and comparable in vitro/in vivo version platforms can be utilized to discover the motives of autoimmunity, in addition to how top it can be regulated. there are ways to evaluate immunological and biochemical pathways proper for pathogenesis and to set up and investigate a number of 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)
4. The supernatants are collected, and IL-2 is measured using 100 µL of the culture supernatant in triplicates by Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). 5. The levels of interferon-a and IL-4 or other cytokines in culture supernatants are measured by ELISA using minikits (Endogen, Cambridge, MA). 6. The absorbance of the color reactions is read at 450 nm in an ELISA microplate reader (Bio-Rad). 46 Nambiar et al. Fig. 2. Increased phosphorylation of TCR/CD3-mediated phosphorylation of TCR c-chain with faster kinetics in SLE T cells.
For multiple samples, a master mix should be prepared excluding the RNA, which has to be added at the end. 3. Incubate the samples at 45°C for 15–60 min. 4. Heat the samples at 95°C for 5 min and immediately cool on ice. The singlestranded cyclic DNA can be stored at –80°C if necessary. 4. POLYMERASE CHAIN REACTION 1. 5 units); add 2 µL of the reverse transcription product. 2. Place a drop of paraffin oil on the top. 3. Set up PCR conditions in a Biometra thermocycler as follows: 94°C denaturation for 5 min, followed by 36 cycles at 94°C for 1 min, 67°C annealing for 1 min, 72°C extension for 2 min, and a final extension at 72°C for 7 min.
2. 3. 4. 5. Ethidium bromide (Sigma Aldrich). Seakem gametic technology grade (GTG) agarose (FMC BioProducts, Rockland, ME). ). PCR beads (Amersham Pharmacia, Piscataway, NJ). X-gal (Sigma-Aldrich). 5. Density Gradient Centrifugation and Protein Concentration 1. 2. 3. 4. 5. 6. 7. Sucrose (Bio-Rad, Hercules, CA). Trichloroacetic acid (TCA) (Sigma Aldrich). Sodium chloride (Sigma Aldrich). Morpholino ethane sulfonic acid (MES) (Sigma Aldrich). Brij58 (Sigma Aldrich). Triton X-100 (Fisher Scientific, Suwanee, GA).
Autoimmunity: Methods and Protocols (Methods in Molecular Medicine) by Andras Perl