Intracellular calcium levels were monitored using the calcium-activated photoprotein, aequorin, as described previously [31 (link)]. In brief, HLMVECs were transduced with an adenovirus encoding aequorin at 30MOI, and 24 h later the aequorin was reconstituted by treating cells with 5 μM coelenterazine (Sigma, St. Louis, MO) for 30 min in serum-free and phenol free DMEM. The cells were then exposed to different concentrations of LLO, and luminescence was recorded using a PolarSTAR luminometer (BMG Labtech, Cary, NC).
Polarstar luminometer
Manufactured by BMG Labtech
The PolarSTAR luminometer is a versatile laboratory instrument designed for the measurement of luminescent signals. Its core function is to detect and quantify light emission from various luminescent samples, such as those used in biochemical assays and cell-based experiments. The PolarSTAR provides precise and reliable luminescence measurements, enabling researchers to gather data for their scientific investigations.
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4 protocols using polarstar luminometer
1
Aequorin-based Intracellular Calcium Assay
2
Measuring Intracellular Calcium Levels
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Intracellular calcium levels were measured either with using calcium indicator Fluo-4 AM (Molecular Probes) according to the manufacturer’s instructions or with the calcium-activated photoprotein, aequorin, as described previously (15 (link)). In brief, for aequorin-mediated fluorescence measurements COS-7, HULEC5α or Cav-1 KO HULEC5α cells were transduced with adenoviruses encoding aequorin, RFP or Cav-1 at 15 MOI, and 24 h later the aequorin was reconstituted by treating cells with 5 μM coelenterazine (Sigma, St. Louis, MO) for 1 hour in serum-free and phenol free DMEM containing 0.1 mM EDTA. Then, the cells were exposed to different concentrations of PLY in Hank’s Balanced Soult Solution (HBSS) for 10 minutes, and luminescence was recorded using a PolarSTAR luminometer (BMG Labtech, Cary, NC). After the baseline luminescence values were recorded, calcium chloride was injected at a 1.8 mM final concentration to enable the measurement of calcium induced changes in luminescence which reflect calcium influx.
3
Quantifying RhoA Activity in HLMVECs
4
Niacin Modulates Neutrophil Respiratory Burst
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Respiratory burst assay was performed as previously described.29 (link) In brief, human promyelocytic NB4 cells were differentiated into neutrophilic cells by treating with all-trans retinoic acid ATRA (1 µM, Sigma-Aldrich) for 5 days. Cells (5 × 104/well in 96 well plates) were incubated in phenol-free medium containing L-012 for 10 min, and phorbol 12-myristate 13-acetate (PMA, 100 nM) was applied in the presence or absence of niacin (50 µM or 300 µM). Luminescence was quantified using a POLARstar luminometer (BMG Labtech).
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