At desired time points, 0.9 mL culture was harvested by centrifugation. Cell pellets were immediately frozen in liquid nitrogen and stored at −80°C until used in ATP assays. For ATP measurements, frozen samples were resuspended with 1 mL 1% trichloroacetic acid, and incubated for about 20 min at 30 °C until cells were lysed. Cell lysate was centrifuged at maximum speed in a microcentrifuge for 2 min. The supernatant (about 500 μL) was collected, and the pH of the supernatant was adjusted to ~7.8 with 10M KOH and 25 μL of 200 mM Tris-Base. ATP in the supernatant was determined with an ATP bioluminescent assay kit according to instructions from the manufacturer (Sigma-Aldrich, FLAA-1KT).
Flaa 1kt
Manufactured by Merck Group
Sourced in United States
The FLAA-1KT is a laboratory equipment product offered by Merck Group. It is a fluorescence-based analytical tool used for various applications in scientific research and analysis. The core function of the FLAA-1KT is to provide accurate and reliable fluorescence measurements, but a detailed description of its intended use is not available while maintaining an unbiased and factual approach.
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4 protocols using flaa 1kt
1
Quantitative ATP Assay Protocol
2
Measuring ATP Release from Macrophages
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Macrophages were cultured for 7 days, and then macrophages were plated on poly‐L‐lysine‐coated 12‐mm diameter coverslips and used 48 h later. Macrophages were stimulated for 3 h with Poly(I:C) (50 μg/ml) and LPS (100 ng/ml) in the presence of the ATPase inhibitor ARL67156 (1 mM). Control macrophages were treated with 1 mM ARL67156 only. ATP release from macrophages was measured as we described previously for taste bud and N2a cells (Taruno et al, 2013 (link)). One hundred microliter medium was collected from each well and added to an equal volume of ATP assay solution FLAA‐1KT (Sigma‐Aldrich) to determine ATP concentration by the luciferin‐luciferase assay with a microplate luminometer (Synergy 2, BioTek). The bath solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 5 Na‐pyruvate and 10 HEPES, pH 7.4 with NaOH.
3
Taste-Evoked ATP Release from Taste Buds
4
Confocal Microscopy Analysis of Electroporated Cancer Cells
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To prepare the cancer cells for observation with the confocal microscope, the cells were electroporated in DMEM under various conditions in a 4 mm spaced cuvette type electrode [38 (link),39 (link)]. A coverslip (25 mm) was placed on a 6 well plate, and 1 × 106 cells/well were incubated for 24 h. The coverslips were washed with PBS and then fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. To improve the intracellular permittivity of Phalloidin, 0.1% Triton X-100 was treated with cells for 5 min, and Phalloidin (A12381, Invitrogen, CA, USA) and Calreticulin (CRT, NBP1-47518AF488, Novus, Centennial, CO, USA) were stained for 20 min and 1 h, respectively. The DAPI solution was dropped on the cells, and the coverslip was mounted on the slide. Samples were imaged using an LSM 880 microscope (Zeiss, Carl Zeiss AG, Oberkochen, Germany) and the images were quantitatively analyzed using Image J software.
The supernatant obtained after treatment with IRE on the cells was quantified using ELISA for HMGB1 (NBP2-62767, Novus, CO, USA) and ATP (FLAA-1KT, Sigma, St. Louis, MO, USA) expression levels. The analysis was performed according to the manufacturer’s instructions.
The supernatant obtained after treatment with IRE on the cells was quantified using ELISA for HMGB1 (NBP2-62767, Novus, CO, USA) and ATP (FLAA-1KT, Sigma, St. Louis, MO, USA) expression levels. The analysis was performed according to the manufacturer’s instructions.
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