For each neural structure evaluated, tissue punches obtained from individual subjects were pooled within treatment groups. Aliquots of pooled sample lysates were homogenized (Model Q55 QSONICA, QSonica, LLC, Newtown, CT; 30 sec.) on ice, heated to 95 °C (10 min.) for ATP extraction, and centrifuged (8 × 1000 rpm; 1 min.). Tissue ATP content was measured using ATP Bioluminescent Assay Kit luciferin–luciferase system reagents, as described by the manufacturer (prod. no. FLAA; Sigma Aldrich Inc., MO). Briefly, ATP standard was diluted with sterile water for preparation of standard curve (2.0 −0.00390625 μmole/mL) by serial dilution. ATP Assay mix (50 μL) was pipetted into individual extraction plate wells; after 3 min., 50 μL standard or sample aliquots were added for incubation. Emitted light per well was measured using a Synergy HT microplate luminometer (BioTek Instruments, Inc., Winooski, VT).
Synergy ht microplate luminometer
Manufactured by Agilent Technologies
The Synergy HT microplate luminometer is a versatile laboratory instrument used for the detection and quantification of luminescent signals in microplate-based assays. It is designed to measure bioluminescence, chemiluminescence, and other luminescent signals with high sensitivity and accuracy.
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Lab products found in correlation
2 protocols using synergy ht microplate luminometer
1
Tissue ATP Content Quantification
2
Comparative Analysis of AAV Vectors
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Infection of HEK293 cells with AAV2, AAV3b, and AAVv66 vectors packaged with CB6-Fluc were performed in biological triplicates. Cells were cultured in Dulbecco’s Modified Eagle Media (DMEM/High Glucose) (Hyclone, SH30022LS) with 10% fetal bovine serum (FBS, Hyclone) under standard cell culture conditions. Cells were lysed 48 h post-infection to assess the infectivity of vectors via luciferase activity using the Luciferase Assay System, (Promega, E1501) and measurements were performed on a Synergy HT microplate luminometer (BioTek, Winooski, VT). Values are expressed as relative light units.
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