Fura-2 AM imaging protocol

Last edited Fri 16 Oct 2015

This protocol has been successfully adapted for different cell types, including fibroblasts, PC12 cells, and embryonic neurons, with variations in cell density and Fura-2 AM concentration.

Stage 1 - Reagent preparation

Materials required

• Fura-2 AM (for example ab120873)
• HBSS-1X (see recipe below)
• 50 mM KCl (see recipe below)

Steps

Mix Fura-2 AM with DMSO.

• Add 50 µL of DMSO to 50 µg of lyophilized Fura-2 AM and vortex for 1 min.

Prepare Hank’s Buffered Salt Solution 10X (HBSS-10X).

• For 1000 mL, use 850 mL of distilled H₂0 (18 Mohm) and add the reagents from the table below, stirring.
• Bring to 1000 mL final volume.
• Store at 4 ^oC.

Prepare HBSS-1x solution.

• Mix 100 mL of the 10X HBSS solution (prepared in the previous step) with 800 mL 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.

Prepare 50mM KCl solution.

• 1 liter volume, pH 7.35, ~290-310 mOsm. Store 4 ^oC.

Stage 2 - Replating

Steps

One to two days prior to experimentation, replate cells to collagen coated coverslips (round, glass, sterilized with ethanol, dried) placed in 35 mm tissue culture dishes.

Place 35 mm dishes in 150 mm Petri dishes that will be used as a microincubator and carrier between incubator and experiments.

When replating, place cells in center of round glass coverslips to settle and ensure that imaging is optimized.

Empirically determine the density of cells and how to replate to have cells stick to glass through the washes and the perfusion of solutions.

Stage 3 - On the day of FURA-2 AM imaging

Materials required

• Fura-2 AM (for example ab120873)
• Ionomycin, free acid (for example ab120370)
• HBSS-1X

Steps

Take the HBSS and 50 mM K solutions out of the refrigerator, turn on equipment and perform calibration curve.

• Check that cells are well adhered to the glass.

Take two 50 mL falcon tubes and label as HBSS and HBSS+BSA.

• Pour HBSS from the HBSS container into the 50 mL tube labeled as HBSS.

Prepare HBSS+BSA solution.

• Measure ~45 mg of BSA (Fatty acid free) and add to the empty tube labeled HBSS+BSA.
• Transfer ~45 mL of HBSS to the same tube and mix gently so as not to create a lot of bubbles, but to mix the BSA completely, then let it settle.
• This should be a 1 mg/mL mixture of BSA and HBSS.

Mix Fura-2 and HBSS+BSA.

• Thaw the Fura-2 AM in 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 the 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 (a 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 them on the bench, then vortex the 15 mL tube on high for 1 min.
• Set in a rack with the 2 x 50 mL tubes of HBSS and HBSS+BSA (loosen/remove lids to all Falcon tubes).

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 it 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 not to cause disruption of plated cells.
• Pull up the same 1 mL of HBSS and eject it into the waste container, bringing up the next 1 mL of HBSS with the same tip and placing gently on the cells.
• Remove the solution again and discard the tip.
• With a new sterile pipette tip, pipette up 1 mL of HBSS+BSA and wash gently, removing waste.
• With the same pipette tip, repeat two 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 the CO₂/37^oC incubator for the 45 min loading time.

Wash the cells.

• 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 3-4 times with 1 mL of HBSS (not HBSS+BSA) each time gently, and place 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 it in the incubator for 30 min-45 min.

Perform imaging.

• 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.
• Constantly perfuse the cells with HBSS on the recording chamber for resting Ca²⁺ measurements. To stimulate cells, 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 two coverslips of cells loading about the same time as washing the first sets.

• Keep track of loading/washing, and know how long it takes to switch coverslips and do the imaging throughout so you can keep to time.