Methods 31 Real Time PCR

After informed consent, heparinized blood (in 10-mL green-top tubes) is obtained by venipuncture from each individual. 1. Blood is mixed with an equal amount of HBSS and layered carefully over 12 mL of Ficoll-Paque Plus in a 50-mL Falcon tube. Spin at 700g for 20 min with brakes off (gradient centrifugation). 2. With a Pasteur pipet, remove carefully the PBMC cell layer from the interface between Ficoll and diluted plasma. Try to avoid obtaining too much Ficoll. Spin at 300g for 10 min. 3. Wash...

Utilization of Tetramers in the Study of Autoreactive T Cells

Autoantibody assays in radioimmunoassay formats have demonstrated disease specificity and sensitivity (e.g., IAAs) for the NOD mouse model. Although it has traditionally been easier to study the humoral component of the immune system, the development of tetramers to study antigen-specific CD4+ and CD8+ T cells has led to improved studies of cellular immunity in type 1 diabetes. MHC-peptide tetramers are soluble complexes of major histocompatibility complex (MHC) molecules bound to streptavidin...

Gradi Frac Control Settings

Control unit UV-1 Knob set to 0.5, switch set to range AU. 2. Pump P-50 Flow rate adjustments will be made using the control panel on the fraction collector. 3. Chart recorder Chart speed set to 5 mm min, blue pen range set to 10 mV, and red pen range set to 1 V. For operation, the valve settings of the GradiFrac are shown in Fig. 1. Fig. 1. Valve settings of the GradiFrac. Fig. 1. Valve settings of the GradiFrac. Fig. 2. Schematic representation of the tubing connection on the main port of the...

Application of Linkage to SLE

There are several different study designs with variable ascertainment approaches that have been used for genomewide scan to identify novel susceptibility loci for SLE. Some of the study designs involve (a) sibling pairs who may or may not have parents available or (b) small and large pedigrees with several generations. Numerous genome scans have been made by the four major scientific groups (located in California, Oklahoma, and Minnesota in the United States and in Sweden), revealing many loci...

Methods for Inducing Apoptosis

Roberts, Antony Rosen, and Livia A. Casciola-Rosen Apoptotic cells are sources of tolerogenic material during tissue homeostasis abnormalities in apoptosis or in the clearance of apoptotic material generate a novel source of antigens against which an autoimmune response may be initiated. In our laboratory, we study the biochemistry and cell biology of systemic autoimmune disease autoantigens during different forms of cell death. Several different methods for inducing apoptosis, and...

Measurement of Intracellular ATP and ADP Levels

T-cell activation and apoptosis require the energy provided by ATP (77). Intracellular ATP concentration is a key switch in the cell's decision to die via apoptosis or necrosis (39) therefore, depletion of ATP may be responsible for defective apoptosis and a predisposition to necrosis in patients with SLE (36). Intracellular ATP levels can be determined with great sensitivity and specificity using the luciferin-luciferase method (78). Addition of luciferin and firefly luciferase to an...

Mitochondrial Hyperpolarization and ATP Depletion Predispose Lupus T Cells to Necrosis

In response to treatment with exogenous H2O2, a precursor, or ROI, lupus T cells failed to undergo apoptosis, and cell death preferentially occurred via necrosis. As noted in ref. 24, H2O2 triggered a rapid increase of AWm and ROI production, which was followed by apoptosis of PBLs in healthy subjects. By contrast, H2O2 failed to elevate AWm, ROI production, and apoptosis, but rather elicited necrosis in patients with lupus. Both CD3 CD28-induced H2O2 production and H2O2-induced apoptosis...