Autoimmunity: Methods and Protocols by Andras Perl

By Andras Perl

This selection of effectively reproducible molecular ideas and similar in vitro/in vivo version structures can be utilized to discover the motives of autoimmunity, in addition to how most sensible it can be regulated. there are ways to evaluate immunological and biochemical pathways suitable for pathogenesis and to set up and verify quite a few autoimmune ailments, together with arthritis, lupus, diabetes, a number of sclerosis, myocarditis, thyroiditis, scleroderma, uveitis, and vitiligo.

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6. Incubate the cells at 4°C for 1 h in a shaker in the cold room. 7. Centrifuge the cells at 18,000g for 10 min at 4°C and collect the supernatant. The pellet, which can be used to evaluate tyrosine phosphorylation of proteins in detergent-insoluble membrane fractions, should be washed twice with 100 µL lysis buffer. 8. Freeze both the pellet and the cytoplasmic protein extract at –80°C. Abnormal SLE T-Cell Signaling 41 Fig. 1. TCR/CD3-induced intracellular calcium response in SLE T cells. For this, 1 × 106 SLE T cells were resuspended in 1 mL prewarmed culture medium and loaded with 1 µL (1 µg/mL in DMSO) INDO-1 (Molecular Probes) for 60 min at 37°C.

Hum. Genet. 44, 388–396. 12. 12 Litt, M. and Luty, J. A. (1989) A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44, 397–401. 13. 13 Lindqvist, A. , et al. (2000) A susceptibility locus for human systematic lupus erythematosus (hSLE1) on chromosome 2q. J. Autoimmun. 14, 169–178. 14. , et al. (2202) A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans.

The methods and protocols described here pertaining to T-cell signaling abnormalities in SLE T cells are very much optimized in many ways, and they were derived by the combined tasks and continuous efforts of many researchers in the laboratory over a long period. These simplified protocols can be readily applied to study T-cell signaling abnormalities in SLE to identify the genetic, molecular, and biochemical factors contributing to aberrant immune cell function and unravel the pathophysiology of SLE.

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