1. Resuspend approximately 5 million splenic cells or purified B cells in 250 ul media (DMEM + 10 mM HEPES and 2.5% FBS). This amount of cells may be sufficient for multiple conditions/stimulations. You may need to adjust this number depending on your experiment.
2. Thaw Fura Red and Fluo-4. Thaw Pluronic acid F-127 (optional).
Note: The two dyes above are labeled as cell permeant and therefore do not really need Pluronic acid. However, the use of pluronic acid has been reported to facilitate dye entry, eliminating any possible hydrolysis of the dyes by external esterases, and more efficient loading while maintaining cell integrity. I’ve used this protocol successfully with and without pluronic acid.
Note: The Fura Red (Invitrogen/Molecular Probes # F-3020 MW = 1089.00) emits at 685 nm (PerCPCy5.5 channel). Its signal will go down when it binds calcium. The Fluo-4 (Invitrogen/Molecular Probes #F14217 MW = 1096.95) emits at 525 nm (FITC channel). Its signal will go up when it binds calcium. Adjust the intensity for each channel so that there will be room in the axes to see these shifts. The starting intensities of the two channels should be close to each other so that baseline ratio will approximate 1.
3. Prepare a 2x strength mixture of these dyes in media. Make enough volume for the number of cell samples you want to stimulate.
Fura Red final concentration (final concentrations I’ve used: 5 – 20 uM; 6 uM final concentration is good.)
Fluo-4 (final concentrations I’ve used: 2.4 to 5.0 uM; 3 uM final concentration is good.)
Pluronic acid (final of 0.01 to 0.02% recommended)
Note: The fluorescence of Fura Red tends to be weaker than Fluo 4, so it is recommended to use a higher concentration of Fura Red compared to Fluo 4 (e.g. 2:1).
Optional: You can also prepare samples with single dyes for compensation.
4. Add 250 ul of 2x dye mix to 250 ul cell suspension. Mix.
5. Incubate cells at 37°C, 45 min in the dark (loading). (You can place tubes in a heat block set at 37°C. Cover the tubes with foil.)
6. Wash once with media, spin and resuspend in 500 ul media.
7. Let the loaded cells rest for 20 min at RT in the dark.
8. If needed (for example, if your starting material is mixed splenic cells), stain with B220 APC (or other conjugates with fluorochromes other than FITC and PerCP Cy5.5). Resuspend cells in media plus B220 APC and incubate at RT for 15 min. Wash with media. Resuspend in 500 ul media.
9. Take an aliquot of loaded cells into a tube and dilute with RT media (0.5 to 1ml) so that the suspension is about 1- 4 million cells/ml. Place the cell suspension on the cytometer and acquire. Adjust flow speed (slow or medium speed, about 400 to 2000 cells/second). Set the number of events to collect at a high number such as 1 million so that the machine does not stop collection too early. Adjust the baseline intensities for FITC and PerCPCy5.5 as described in #2.
Keep extra loaded cells in the dark at RT, or if kept on ice, you will need to warm them up to RT before stimulation.
Just before fluxing, make sure that stimulating reagents are ready.
Optional: Compensate using single stains.
10. To flux, acquire and record for 30 seconds. Remove the tube from the cytometer (without stopping the acquisition/recording), quickly add stimulus to cell suspension and quickly mix. Return the tube to the cytometer and continue reading for at least 5 min. You should see a right shoulder forming when viewing the FITC channel.
Note: For BCR stimulations, I use a final of 10 ug/ml of anti-IgM F(ab’)2.
Optional: Stimulate one sample with 1 ug/ml ionomycin to establish maximum flux. Or, stimulate in the presence of a final concentration of 6-8 mM EGTA added in the media just before reading the sample to chelate extracellular calcium.
11. To analyze in FlowJo (v 8.1.0), go to Platform/Derive Parameters/Define new/change. Choose parameter and function to create a new parameter for the ratio of Fluo 4/Fura Red. For example, choose ‘FITC’, choose ‘division’ then choose ‘PerCPCy5.5’. Click ‘Add Parameter’ and then ‘Done’. In the Workspace, select sample, then Click ‘Kinetics’. In the kinetics window, change the y axis to the ratio parameter.