Mitochondria were isolated according to a previous report13 (link) with some modifications (see Suppl. Materials and Methods ). The ADP/ATP exchange assay was carried out in a filter plate format using a 96-well MultiScreenHTS FC filter plate (Millipore, Billerica, MA). Compound or DMSO diluted into mitochondria isolation buffer (MIB) (20 mM HEPES-KOH, 0.6 M mannitol, pH 7.4) was transferred into the precooled plate (50 μL/well). Mitochondria were added in 25 μL MIB (50 μg/well) and incubated for 10 min. ADP/ ATP exchange was initiated by adding 25 μL of 20 μM [3H] ADP (5 μCi/mL). The exchange reaction was then stopped after 1 min by the addition of 25 μL cATR (5 μM final). The reaction mix in the filter plate was then through on a vacuum manifold, and the plate was washed four times with 300 μL/well MIB buffer. Supermix scintillation cocktail (PerkinElmer, Waltham, MA) (100 μL) was transferred into each well and incubated overnight, and the [3H]ADP taken up by mitochondria was measured with a MicroBeta scintillation counter (PerkinElmer).
Supermix scintillation cocktail
Manufactured by PerkinElmer
Supermix is a scintillation cocktail product manufactured by PerkinElmer. It is designed for use in liquid scintillation counting applications to detect and measure radioactive samples.
Automatically generated - may contain errors
2 protocols using supermix scintillation cocktail
1
ADP/ATP Exchange Assay for Mitochondria
2
Quantitative Assay for Autoantibodies
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Recombinant GAD65 and IA-2 were labeled with 35S-methionine (PerkinElmer, Waltham, MA, USA) by in vitro coupled transcription and translation using the TNT SP6 coupled reticulocyte lysate system (Promega, Madison, WI, USA) as described [14 (link)]. Full length cDNA coding for human GAD65 in the pTNT vector (Promega) (pThGAD65) or the intracellular domain (amino acids 606-979) of IA-2 in the pSP64 Poly(A) vector (Promega) (IA-2ic) were used as templates [15 (link)]. GADA and IA-2A were analyzed in a radioligand binding assay (RBA) [14 (link)]. Duplicate samples were incubated with radio-labeled antigen. The samples were transferred to filtration plates (Millipore, Solna, Sweden) and IgG antibodies precipitated with Protein A Sepharose (Zymed Laboratories Inc, San Francisco, CA, USA). After washing to remove all unbound antigen supermix scintillation cocktail (Perkin Elmer) was added and the radioactivity counted in a Wallac Microbeta Trilux system (Perkin Elmer). GADA and IA2A levels were expressed as units per mL (U/mL) derived from the WHO standard 97/550. GADA levels >34 U/mL and IA-2A levels >5 U/ml were considered positive.
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