Spectramax plus

Concentration of serotonin in the saliva is relevant to nociception and the pathogenesis of chronic pain syndromes seen in FM [31 (link)]. Thus, mean serotonin changes from randomization to the end of withdrawal will be evaluated in 25-μl salivary samples by using a competitive enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Human Serotonin ST ELISA Kit; MyBioSource, San Diego, CA, USA). The samples will be collected with cotton swabs into plastic tubes without any stimulation and frozen (−80 °C). Patients will be instructed to rinse their mouths with water and not to eat or drink 30 min before the samples are collected. The ELISA plates will be read with a SpectraMax Plus 384 spectrophotometer (Molecular Dynamics, Sunnyvale, CA, USA) at a wavelength of 400 nm [31 (link)].

HeLa cells transiently transfected with the indicated plasmids were plated in 24-well plates (0.5 x 10⁵ cells/well) and incubated overnight. For siRNA experiments, cells were transfected with plasmid, incubated overnight and then transfected with siRNA. Cells were stimulated with 10 μM ADP for the indicated times, then washed with ice-cold PBS. Cells were then fixed with 4% paraformaldehyde (PFA), washed with PBS and incubated with anti-FLAG antibody for 1 h. Cells were washed and incubated with HRP-conjugated secondary antibody for 1 h at room temperature, then developed using 1-Step ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (Thermo Fisher). The amount of surface P2Y₁ was quantified by determining the absorbance of an aliquot at 405 nm using a Spectramax Plus (Molecular Dynamics) spectrophotometer.

Serum paraproteins were detected in 10 μl of undiluted serum using Hydragel 15 HR electrophoresis kit on a Hydrasys apparatus (Sebia, France) according to the manufacturer’s instructions. Monoclonal bands were detected visually on stained gels.
Paraprotein isotypes in sera or ascitic fluids were determined by ELISA using isotype-specific goat anti-mouse IgM, IgG (IgG1, IgG2a, IgG2b, IgG3), and IgA labeled with horseradish peroxidase (Southern Biotech Associates, Birmingham, AL). Immulon 2 HB plates (Dynex Technologies, Chanitlly, VA) were coated with serum or ascitic fluid at dilutions of 10⁻³ to 1.28 × 10⁻⁵. Plates were read on a microplate reader SpectraMax Plus (Molecular Dynamics, Sunnyvale, CA) at 450 nm and data analyzed using v5.3 SoftmaxPro software of the same company.