Fura-2 AM Staining Protocol
This protocol has been successfully adapted to different cell types, including fibroblasts, PC12 cells, and embryonic neurons, with different Fura-2 AM concentrations corresponding to different cell densities .
Phase 1 Reagent preparation
Materials required
- Fura-2 AM
- HBSS-1× (see recipe below)
- 50mM KCl (see recipe below)
Experimental steps
1. Mix Fura-2 AM and DMSO.
• Add 50 µL DMSO to 50 µg lyophilized Fura-2 AM and vortex for 1 minute.
• Tips: Wrap in foil to protect from light and store at -20℃.
2. Prepare Hank 's buffered saline solution 10× (HBSS-10×).
2.1 For 1000 mL , use 850 mL of distilled water and add the reagents listed in the table below, stir.
2.2 The final volume is set to 1000 mL.
2.3 Store at 4°C.
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3. Prepare HBSS -1× solution.
3.1 Mix 100 mL of 10× HBSS solution (prepared in the previous step) with 800 mL of distilled water.
3.2 Add 0.14 g anhydrous CaCl2 ( MW 111, 1.3 mM), 1 g d-glucose ( MW 180.2, 5.5 mM) and 0.35 g NaHCO3 ( MW 84.01, 4.2 mM).
3.3 Add distilled water to make up to 1000 mL, adjust the pH to 7.4, and store at 4 °C for about 1 week.
4. Prepare 50 mM KCl solution.
• 1L volume, pH 7.35, ~290-310 mOsm. Store at 4°C.
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Phase 2 Replating
Experimental steps
1. One to two days before the experiment, re-seed the cells onto collagen-coated coverslips (round, glass, sterilized with ethanol, dried) placed in 35 mm tissue culture dishes .
2. Place the 35 mm dish into the 150 mm dish and use it as a carrier between the micro-incubator and the incubator and the experiment.
3. When reseeding, center the cells on the round glass coverslip to stabilize them and ensure optimal imaging.
4. Determine empirically the cell density and how to re-seed so that the cells adhere to the glass after washing and perfusion solutions.
Phase 3 FURA-2 AM imaging day
Materials required
- Fura-2 AM
-Ionomycin, free acid
- HBSS-1×
Experimental procedures
1. Take the HBSS and 50 mM KCl solutions out of the refrigerator , turn on the equipment and plot the calibration curve.
• Note: Check that the cells are well adhered to the glass.
2. Take two 50 mL falcon tubes and label them as HBSS and HBSS+BSA respectively.
• Pour HBSS from the HBSS container into the 50 mL tube labeled HBSS.
3. Prepare HBSS+BSA solution .
3.1 Weigh approximately 45 mg of BSA (fatty acid-free) and add to the empty tube labeled HBSS+BSA.
3.2 Transfer about 45 mL of HBSS to the same tube, mix gently so as not to create a lot of bubbles but to mix the BSA thoroughly, and let it settle.
3.3 This should be a 1 mg/mL mixture of BSA and HBSS.
4. Mix Fura-2 and HBSS+BSA .
4.1 Turn off the room light , thaw Fura-2 AM in 50 µl DMSO and mix.
4.2 Only load 2 of the 35 mm dishes at a time, as the timing for imaging will not work well for the timing of the post-Fura wash.
4.3 Pipette 4-10 µL/35 mm dish of Fura-2/HBSS/BSA mixture into a 15 mL Falcon tube (lower Fura-2 AM concentrations in cells used for imaging will reduce the buffering capacity within the cells).
4.4 Add 4 mL of HBSS+BSA to the 15 mL tube containing Fura-2.
4.5 Take out the two coverslips from the incubator, place them on the bench, and vortex the 15 mL tubes at high speed for 1 minute.
4.6 Place it in a rack containing 2×50 mL HBSS and HBSS+BSA tubes (loosen/remove the caps of all Falcon tubes).
• Tip: Keep a waste container nearby and sterile pipette tips open and ready.
5. Load Fura.
5.1 Take out two 35 mm culture dishes from the incubator.
5.2 Using a 1 mL sterile pipette tip, remove all of the culture medium from one 35 mm cell culture dish and drain it into a waste container.
5.3 Using the same pipette tip, take out 1 mL of HBSS and gently add it to the cells, being careful to place it along the side and add gently to avoid disrupting the plated cells.
5.4 Pull out the same 1 mL of HBSS and eject it into the waste container, take out the next 1 mL of HBSS with the same tip and gently place it on the cells.
5.5 Remove the solution again and discard the tip.
5.6 Using a new sterile pipette tip, draw up 1 mL of HBSS+BSA and wash gently to remove waste.
5.7 Using the same pipette tip, repeat twice more and wash the cells 3 times with HBSS + BSA.
5.8 Using the same pipette tip, immediately add 1 mL of Fura+HBSS+BSA that has been vortexed for 1 minute.
5.9 Add the second 1 mL of Fura-2 AM to the cells and mark the time on the cover slip. Repeat the above steps for the second dish.
5.10 Typically, the loading time is 45 minutes, but this may need to be varied, as well as placing 4-10 µL of Fura-2 AM in 2 mL of HBSS+BSA. Return both culture dishes to the CO2/37°C incubator and allow the loading time to be 45 minutes.
• Tip: Keep a waste container nearby and sterile pipette tips open and ready.
6. Wash the cells.
6.1 Take out two culture dishes and use a new sterile pipette tip to gently pipette 1 mL of the Fura-HBSS + BSA mixture into the waste solution, and then pipette a second 1 mL.
6.2 Using a new pipette tip, gently wash the cells 3-4 times with 1 mL of HBSS (not HBSS+BSA) each time and discard the wash solution into waste.
6.3 After the 4th wash, keep 1 mL of HBSS and add a second 1 mL of HBSS. Mark the time and return it to the incubator for 30 to 45 minutes.
7. Perform imaging.
7.1 Place the first cell coverslip in the washer for about 25 minutes before placing it on the imaging device in the chamber.
7.2 Perform the imaging experiment within 7-15 minutes at most and then remove the next dish from the incubator.
7.3 Keep perfusing the cells with HBSS in the recording chamber for resting Ca2+ measurements. To stimulate the cells, use some stimulus, either the test solution, 50 mM KCl solution, or electrical stimulation.
7.4 For stimulation, use a series of high K+ solutions (matched monovalent): 20 mM, 40 mM, and 60 mM KCl solutions, each time replacing the NaCl with an equal amount of KCl.
7.5 To obtain a positive control for imaging, use a 20 µM working concentration of Ionomycin (5 mg free acid mixed to 20 mM in DMSO) and place an appropriate volume into the chamber volume during perfusion.
• Warning: All experiments should be done in the dark.
8.To continue through the day, we recommend beginning the next two coverslips of cells loading about the same time as washing the first sets.
• Keep track of your loading/washing process to understand how long it takes to switch coverslips and perform imaging so you can stay on schedule.
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