Loading Cells with Ca-Sensitive Dyes
- 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.
- 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!)
- Remove media from dish and replace with 1.5ml of Microscopy Buffer for 10 min.
- Add 10µl of dye solution to 1.5ml of Microscopy Buffer in a 15 ml tube and mix gently.
- Replace buffer in dish with the diluted dye (#4 above) for 20min. Keep dish in a foil-covered box.
- Add 1.5 ml of Microscopy Buffer to dish for 10min.
- Rinse dish with 1.5 ml of fresh Microscopy Buffer. Remove buffer and immediately add 0.75ml of fresh Microscopy Buffer.
- 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.
- Position bottom of chamber over coverslip and tighten.
- Fill chamber immediately with 500µl of buffer.
- 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.