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This protocol has been successfully adapted for different cell types, including fibroblasts, PC12 cells and embryonic neurons with variations on cell density and Fura-2 AM concentration.
Mix Fura-2 AM 50 µg lyophilized by adding 50 µL of DMSO and vortex for 1 min. Store at -20 oC wrapped in foil for light protection.
Mix Hank’s Buffered Salt Solution (HBSS-10X)-with final 1x concentration:
|NaCl (FW 58.44)||80 g (137 mM)|
|KCl (FW 74.55)||4 g (5.4 mM)|
|MgCl2-6H20 (FW 203.3)||1g (0.5 mM)|
|MgSO4-7H20 (FW 246.5)||1g (0.4 mM)|
|KH2PO4 (FW 136.1)||0.6 g (0.44 mM)|
|Na2HPO4-7H20 (FW 268.1)||0.9 g (0.34 mM)|
For 1000 ml, use 850 mLs distilled H20 (18 Mohm), add in order, stirring. Bring to 1000 mL final volume. Store at 4 oC.
Mix HBSS-1x solution:
Mix 100 mLs of the 10X HBSS solution with 800 mLs of distilled H20. Add 0.14 g of anhydrous CaCl2 (FW 111, 1.3 mM), 1g of d-glucose (FW 180.2, 5.5 mM) and 0.35 g NaHCO3 (FW 84.01, 4.2 mM). Bring to 1000 mL volume with distilled H20, pH to 7.4 and store for ~1 week at 4 oC.
Mix 50 mM KCl:
|NaCl (FW 58.44)||5.0843 g (87 mM)|
|KCl (FW 74.56)||3.728 g (50 mM)|
|MgCl2 (FW 203.3)||0.2033 g (1 mM)|
|CaCl2 (1 M Stock from certified volumetric stock)||5 ml (5 mM)|
|HEPES (FW 238.3)||2.833 g (~12 mM)|
|Glucose (FW 180.16)||1.8016 g (10 mM)|
1 liter volume, pH 7.35, ~290-310 mOsm. Store 4 oC.
Replating: One to two days prior to experimentation, replate cells to collagen coated coverslips (see protocol below, round, glass, sterilized with ethanol, dried) placed in 35 mm tissue culture dishes. The 35 mm dishes are placed in 150 mm Petri dishes to be used as a microincubator and carrier between incubator and experiments.
When replating, place cells in center of round glass coverslips to settle and to ensure that imaging is optimized. Empirically determine the density of cells and how to replate in order to have cells stick to glass through the washes and the perfusion of solutions.
On day of experiment:
Take the HBSS and 50 mM K solutions out of the refrigerator, turn on equipment, perform calibration curve, check that cells are well adhered to glass.
Take 2 x 50 ml Falcon tubes, label as HBSS and HBSS+BSA. Pour HBSS from the HBSS container into the 50 ml tube labeled as HBSS.
Mix HBSS+BSA: Measure out ~45 mg of BSA (Fatty acid free) and add to the empty tube labeled HBSS + BSA. Transfer ~45 ml of HBSS and mix gently so as not to create a lot of bubbles, but to mix the BSA completely, let settle. This should be a 1 mg/mL mixture of BSA and HBSS.
Mix Fura-2 + HBSS+BSA: Thaw the Fura-2 AM 50 µl DMSO and mix with room lights turned off. Only load 2 of the 35 mm dishes at a time, as the timing for imaging will not work well for timing of the post-Fura wash. Pipette 4-10 µL/35 mm dish of cells of this Fura-2/HBSS/BSA mixture into a 15 mL Falcon tube (lower concentration of Fura-2 AM in the cells for imaging will reduce the intracellular buffering capacity). Add 4 mL of HBSS+BSA into the 15 mL tube with Fura-2. Get two dishes of coverslips from the incubator, place on the bench, then vortex the 15 mL tube on high for 1 min. Set in rack with the 2 x 50 mL tubes of HBSS and HBSS+BSA (loosen/remove lids to all Falcon tubes). Have a waste container nearby and sterile pipette tips open and ready.
Load Fura: Remove 2 of the 35 mm dishes from the incubator. With 1 mL sterile pipette tips, remove all media from one 35 mm dish of cells and eject into a waste container. With the same tip, bring up 1 mL of HBSS and gently add to the cells, being careful to place along the side and gently to not cause disruption of plated cells. Pull up the same 1 mL of HBSS and eject into the waste container, bringing up the next 1 mL of HBSS with the same tip and placing gently on the cells. Remove solution again, and discard tip. With a new sterile pipette tip, pipette up 1 mL of HBSS+BSA and wash gently, removing to waste. With the same pipette tip, repeat 2 more times to wash the cells 3x with HBSS + BSA. With the same tip, immediately add 1 mL of the Fura +HBSS+BSA that was vortexed for 1 min. Add the second 1 mL of Fura-2 AM to the cells and label the lid of the coverslip dish with the time. Repeat for the second dish.
Typically, load for 45 min, but this might need to be varied as well as the 4-10 µL of Fura-2 AM in 2 mL of HBSS+BSA. Replace both dishes in CO2/37 oC incubator for the 45 min loading time.
Wash: Remove both dishes and gently, with a new sterile tip, pull 1 mL of the Fura-HBSS+BSA mix to waste, and then the second 1 mL. With a new pipette tip, wash the cells 3-4x with 1 mL of HBSS (not HBSS+BSA) each time gently, and placing the wash in the waste. After the 4th wash, leave on 1 mL of HBSS and add a second mL of HBSS. Label the time and replace in the incubator for 30 min-45 min.
Get the first coverslip of cells about 25 min into the wash and set up on the imaging rig in the chamber. Perform the imaging experiment in 7-15 min maximum, and then get the next dish from the incubator. All experiments should be completed in the dark.
Constantly perfuse the cells with HBSS on the recording chamber for resting Ca2+ measurements and to stimulate use a stimulant of some sort, either the test solution, or 50 mM K solution, or electrical stimulation. To stimulate use a range of high K+ solutions (matched monovalents): 20 mM, 40 mM and 60 mM KCl solutions as well, each time replacing NaCl with equivalent amounts of KCl.
To obtain a positive control for imaging, use 20 µM working concentration ionomycin (ab120370, 5 mg free acid mixed to 20 mM in DMSO) by placing the appropriate volume into the volume of the chamber while perfusing.
To continue through the day, we recommend beginning the next 2 coverslips of cells loading about the same time as washing the first sets, keeping track of loading/washing and knowing how long it takes to switch coverslips and do the imaging experiments.