Measurement of calcium flux on the FACSVantage DiVa using indo-1.
The ratiometric calcium probe indo-1 (AM ester form) can be loaded into cells for analysis of calcium flux following cell stimulation. Detection of the ratio between bound indo-1 and free indo-1 (and hence the relative intracellular calcium concentration) can be carried out on the FACSVantage using a UV laser source for indo-1 excitation and 390/20 and 495/20 nm narrow bandpass filters for detecting bound and free indo-1 fluorescence respectively.
We use our krypton-ion laser in the tertiary position tuned to muliline UV output (100 mW) as an excitation source. The free and bound forms of indo-1 are then detected in the FL10 and FL11 PMTs on our upgraded 11-color optical bench using the above bandpass filters and a 460 longpass dichroic with extended transmission to split the signals. All other dichroics in the signal path are removed. Alignment is carried out using Hoechst 33342-labeled chicken erythrocytes or Polyscience yellow-green 2 um beads. We use a Cytek Time Zero kinetic module to add reagents to cells on-line.
(Below). Optical bench configuration for detection of indo-1 using the tertiary beam path. Indo-1 is excited with the tertiary krypton-ion laser tuned to multiline UV. 390/20 and 495/20 nm narrow bandpass filters (custom-fabricated by Chroma Technology) are used to detect bound and free indo-1 respectively, the two signals split by a 460 longpass dichroic (fabricated with extended transmission to the 390 nm range).
(Below). Our Cytek Time Zero kinetic module set up on our FACSVantage SE. We use the instruments air supply to provide sample chamber normal and boost air.
(Below). Human monocytes loaded with indo-1 AM (Molecular Probes) at 3 uM at 37 degrees centigrade for 30 minutes and analyzed on the FACSVantage using the above filter configuration. Ionomycin at 1 µM was added to the cell population on-line using the Cytek Time Zero kinetics module at 30 seconds following the start of data acquisition. The dotplots on the left show the fluorescence emission at 390 nm (bound indo-1) and 495 nm (free indo-1). Both wavelengths were collected in linear scale. The plot on the right shows the ratio between 390 and 495 nm.
(Below). Murine EL4 cells were loaded with indo-1 AM at 5 uM at 37 degrees centigrade for 30 minutes and analyzed on the FACSVantage using the above filter configuration. Ionomycin at 1 µM was added to the cell population on-line using the Cytek Time Zero kinetics module at 30 seconds following the start of data acquisition. The dotplots on the left show the fluorescence emission at 390 nm (bound indo-1), 495 nm (free indo-1) and the ratio between the two wavelengths. Both wavelengths were collected in linear scale. The plot on the right shows the ratio between 390 and 490 nm.
Excitation of indo-1 off the krypton tertiary laser allows us to use the primary laser to excite one or more PMTs. FITC is difficult to use with indo-1 since their emission spectra overlap, resulting in signal crosstalk; however, PE, PE-Cy5 and APC can all be used as immunolabeling fluorochromes simultaneously with indo-1 (APC off the HeNe 633 nm beam). Phenotype-specific calcium flux can then be measured.
(Below). Murine EL4.153.1 (CD153 sort-selected) cells were loaded with indo-1 at 5 uM at 37 degrees centigrade for 30 minutes, washed and subsequently immunlabeled with PE-anti-CD153 (Caltag) for 20 minutes. The cells were then analyzed on the FACSVantage using the above filter configuration. The histogram on the left shows the PE-CD153 fluorescence, being positive for the entire population but varying from low to high. The 3-D dotplots (middle, isotype control; right, PE-CD153) show PE-CD153 expression on the X-axis, and bound and free indo-1signals on the Z- and Y-axes respectively). Addition of ionomycin results in a signal shift from free to bound indo-1; calcium flux in CD153 low (blue) and high (green) subpopulations is clearly visible.
Click here to see indo-1 done on the BD LSR, a benchtop flow cytometer equipped with a HeCad UV laser.