Real-Time calcium transient measurement in mouse dendritic cells stimulated with LPS or ATP.
Lab/Group: Granucci lab (University of Milano-Bicocca)
Related Journal & Article Information
Journal: Nature
Article Title: CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation
Introduction
Protocol for calcium transients measurement in dendritic cells used in our Nature paper.
Intracellular calcium concentration was determined by a fluorimetric ratio technique, an approach that overcomes possible problems of uncertainty related to the calibration or uneven distribution of fluorescent calcium indicators. A direct optical microscope (Olympus, BX51) with a two-photon Ti-Sapph laser source (720 nm wavelength; Mai Tai, SpectraPhysics) was used for indo-1 excitation. The fluorescence signals emitted by indo-1-loaded cells were digitized at 200 Hz and recorded every 0.5-0.8 s. The ratio of fluorescence emissions at 400 nm/bp to those at 500 nm/bp was recorded (R400/500) and used as an index of intracellular calcium concentration. Data were then normalized to baseline.
In some cases, cells were analyzed in calcium-free PBS or calcium-free PBS supplemented with 50 nM thapsigargin (Sigma Aldrich).
Materials
Reagents
Equipment
Procedure
1.Seed 3 × 106 cells (BMDCs or BMMΦs) in 60 × 15 mm non-treated cell culture dishes at a concentration of 106 cells/ml in the appropriate medium.
2.Incubate overnight (37 °C – 5% CO2).
3.Gently wash cells three times with 3 ml of pre-warmed PBS to remove residual extracellular esterases contained in the medium.
4.Add 1 ml of 2 microM of the ratiometric probe indo-1 (Molecular Probes, Eugene, OR) in PBS and incubate 20’ (37 °C – 5% CO2) in the dark.
5.Gently wash cells three times with 3 ml of pre-warmed PBS to remove extracellular free probe.
6.Add 1,8 ml of pre-warmed PBS.
7:Start real-time imaging acquisition and superfuse the appropriate stimulus in 200 μl PBS.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
Dendritic cells, D1 cells, LPS, calcium measurements.