Loading Cells with Ca-Sensitive Dyes

1. Remove an aliquot of the AM form of the calcium sensitive dye (fura, fluo, rhod...) from the freezer and let it come to room temperature for ~10 min.
2. Add 50 µl of 20% Pluronic in DMSO (see below) to the tube and wait ~5min. Draw the solution gently up and down to mix, being careful not to introduce air (Pluronic will cause foaming!)
3. Remove media from dish and replace with 1.5ml of Microscopy Buffer for 10 min.
4. Add 10µl of dye solution to 1.5ml of Microscopy Buffer in a 15 ml tube and mix gently.
5. Replace buffer in dish with the diluted dye (#4 above) for 20min. Keep dish in a foil-covered box.
6. Add 1.5 ml of Microscopy Buffer to dish for 10min.
7. Rinse dish with 1.5 ml of fresh Microscopy Buffer. Remove buffer and immediately add 0.75ml of fresh Microscopy Buffer.
8. Remove the coverslip from dish and mount onto chamber, cells upward. Center the coverslip over the O- ring, then press gently in center of coverslip with tip of forceps to form a seal with the O-ring.
9. Position bottom of chamber over coverslip and tighten.
10. Fill chamber immediately with 500µl of buffer.
11. Wipe the bottom of the coverslip to dry. Check for leaks with a dry piece of tissue for at least 5 minutes!! If no leaks are evident, wipe the coverslip with a cotton swab wetted with glass cleaner and dry. If coverslip is leaking, check for cracks. If leak persists, disassemble chamber and reposition coverslip. Reassemble and check again.

20% weight/volume Pluronic in DMSO

• Weigh 0.2g Pluronic into the bottom of brown glass vial.
• Add 1ml of anhydrous DMSO directly onto the Pluronic.
• Warm to dissolve. Do not vortex until completely dissolved!

Ordering information: Pluronic F-127 from Invitrogen, cat# P-6867.