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Making Ringer's Solution

 

Ringer's Solution is a pH-balanced salt solution that is used for live cell imaging.

From Gerbino, Ruder et al., J Cell Biol. 171 (2), 2005: link to jcb.org

 

 

"Ringer's solution containing 121 mM NaCl, 2.4 mM K₂HPO₄, 0.4 mM KH₂PO₄, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM glucose, and 10 mM Hepes/NaOH, pH 7.40."

 

Making 10X Stock

Warren generally maintains a bottle of ultra-precise 10x Ringer's stock solution which includes:

 

 

1.21 M NaCl (for 500 mL of solution, use 35.3562 g)

24.0 mM K₂HPO₄ (500 mL use 2.0902 g)

4.0 mM KH₂PO₄ (500 mL use 0.27218 g)

100 mM HEPES ( 500 mL use 11.915 g)

 

Making 1X Solution

500 mL batch

You need:

 

 

Clean Glassware

1. 1000 mL beaker for preparation
2. 50 mL precision flask (for measuring 10X stock)
3. 500 mL precision flask (for final dilution)
4. magnetic stirrer.
5. funnel.
6. A clean container to store the solution in.

Also a clean powder scoop, a few transfer pipets, a couple 1 mL or 5 mL pipets.

 

 

pH meter and pH titrating solutions (generally NaOH at 10 M, 1 M, 0.1 M)

 

Mixing the solution

1. Wash glassware and rinse 5X with tap water, 5X with distilled water.
2. Warm the 10X stock in the water bath (it changes density with temperature).
3. Prepare about 150 mL of DI water in the 1000 mL beaker. Place it on the hot plate / stirrer with a magnetic stirrer inside.
4. turn on the stirrer (but not the hot plate).
5. Add 5.5 mM glucose (D-Glucose, anhydrous, 0.496 g or D-glucose monohydrate, 0.545 g).
6. Measure 50.0 mL of 10X stock in the precision flask. Add it to the beaker. Rinse the flask with DI water and empty into the beaker (you don't want to lose ANY of the 50.0 mL).
7. Add 1-1.2 mM MgCl₂ or MgSO₄ (500-600 uL from 1M stock).
8. If desired, add 1-1.2 mM CaCl₂ (500-600 uL from 1M stock).

    

    

    note: obviously, if you are making Calcium-free Ringer's solution, don't add CaCl₂.

9. Add DI water up to about 300-350 mL.

 

Balancing the pH

1. Calibrate the pH meter using the pH 4, pH 7, and pH 10.01 buffers.
   •  Be sure to thoroughly rinse the pH meter with DI water after each stage into a waste container.
2. Submerge the calibrated probe into the 1000 mL beaker. It should probably be around 6-6.5 pH.
3. Add drops of NaOH to increase the pH.
   1. One drop of 10M solution will tend to raise it about 0.1-0.5 pH
   2. One drop of 1M solution will tend to raise it about 0.05-0.1 pH
   3. One drop of 0.1M solution will tend to raise it about 0.005-0.01 pH.

    

    

4. You want a final pH value of 7.4-7.43.
5. Thoroughly rinse the probe into the beaker and put it away.

 

Final Dilution

1. Using the funnel, transfer the contents of the beaker to the 500 mL precision flask.
2. Rinse the beaker with DI water and pour the runoff into the precision flask as well.
3. Add DI water until the total volume is 500 mL.

Pour the contents of the precision flask into the container. Do not rinse the remnants into the container with DI water- this will cause further dilution, which is not desired.

 

Clean up your glassware.

 

 

 

Imaging Dye Loaded Cells

• Turn on the fluorescent lamp and allow it to complete the warm-up cycle.
• Turn on the microscope and the Insight camera.

 

 

Finally, turn on the lambda 10.3 controller and choose appropriate shutter setting (see UsingShutterController).

Set to the 63x objective, ensure that it is thoroughly cleaned then place two drops of lens immersion oil directly onto the lens to create a bubble. Adjust the objective so that it is slightly under the microscope stage.

Place the dye-loaded cells (see additional protocol: LoadingDyeIntoCells) into a stage bracket and clip into the stage manipulator.

Using the stage manipulator, ensure that the microscope lens is placed in the coverslip’s center. Afterwards, slowly ascend the objective until it contacts with the coverslip. The surface tension of the oil on the lens will break when it touches the coverslip.

Using the halogen lamp locate the cells.

On the computer, open the Spot Advance program. Select Live (the fluorescent light should be on- this light should not be on for longer than 5 seconds to avoid photobleaching) and focus the cells quickly until their processes are visible. Press Liveagain to turn fluorescence off.

Select a region that contains cells at the desired confluency and morphology.

Use Live to focus at best on that region. Following that, select the option below Live, Get an image, to take a picture of the region.

Use the picture to determine if additional adjustments are needed. One possible adjustment is the fluorescent intensity (controllable on the lamp power supply). Determine a good range of intensity to first try and focus (the cells should not be too bright and should be focused on the computer). Following the adjustments, take another picture, but ensure that 100% of the light output is directed to the camera

Go to the toolbar, and select Camera, and then Get Sequential Images. For the dialog box that appears, select 240 images for the number of images to take with 5 seconds intervals (or the interval most appropriate for your dye). Also adjust the exposure time (also dye-specific). Finally, in fast mode, you must set the TTL lag so that 8ms passes before the camera exposure begins (when the shutter if fully open). Then create a folder with the movie number and in this folder save this procedure as movie number.

Select Begin.

The shutter and camera will now synchronously take images at the specified intervals and specified exposure time. Make a note of when various stimulants are provided to the cell.

In Image J, open the movie file. Circle the region of interest in the cell. On the toolbar select Analyze, then Tools, and thenROI Manager. Using ROI Manager, select the measure option and the mean value of intensities in the ROI selected will be.

Select measure for the cell and save results as an excel spreadsheet. Make sure to add .xls extension if you will use Excel for data analysis. Repeat this procedure for all the visible cells.

Time traces for each ROI can then be compiled together to plot several cells' ROI responses.

 

The following is a list of live cell dyes used in the Leduc laboratory and good approaches for their use:

Fluo-4-AM

• Prior to the day's experiments, change the medium for the cells.
• Prepare a aliquots of a 1 mM Fluo-4-AM solution in DMSO. Freeze extra aliquots for future use.
• Dilute an aliquot to 2 uM in culture media of the cells.
• Incubate for 20-25 minutes.
• Arrest the loading by changing the cell bath with either fresh media or Ringer's solution for live cell imaging experiments.

 

 

 

This protocol describes use of the Lambda 10-3 controller and computer-controlled Smart Shutter. Unless specifically noted, "shutter" refers to the Smart Shutter, and not the built-in Zeiss shutter.

Before beginning, make sure that the manual (Zeiss) shutter is open. Also, make sure that the fluorescent lamp is already on. You must always turn the shutter controller on after the mercury lamp and turn it off before you turn off the mecury lamp. Turning on the mecury arc lamp power supply produces a surge that can damage the electronics of the shutter controller

To Enable the Controller for Cell Imaging/Movie Making:

• Turn on the device with the off/on switch.

 

 

On the keypad at the right select LOCAL to enable keypad functionality

On the keypad at the right select MODE

Select option 3-TTL

Select 1- Enable shutter (A).

Select 1 –Fast, this determines the speed at which the shutter opens.

Select 0 – No other options

The shutter is now controlled by a TTL pulse from the camera controller card. This allows synchronization of image capture by camera and excitation by lamp

To Disable Controller

 

 

Select MODE.

Choose 3-TTL

Select 2-Disable Shutter (A)

Choose 0-No other options

The shutter is now in manual mode.

 

 

* Press S1 on the keypad until the shutter opens. Ensure the Mercury Arc Light is transmitted through the microscope housing and into the filterblock changer. Turn off the device with the off/ on switch.

Make sure to leave the shutter open for the next user. This will allow users not familiar with the Lambda Smart Shutter to revert back to the built-in Zeiss shutter.

 

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How to use the 63X Objective CAREFULLY 

 

 

 

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Microscope Protocol-LeDuc Lab Inserting the Objective (to eliminate objective damage)

 

1. We will be putting this in a lock-box with personal codes in the future, but we do not have this yet. People need to use this so we are proceeding while we order this box. For now, sign the objective out and remove it from the objective drawer located in the main lab computer desk (not the imaging computer desk).

2. Remove the microscope shroud.

3. Place Styrofoam bucket next to the microscope stage (THIS IS TO PROTECT THE OBJECTIVE IN CASE IT FALLS).

4. Position the 63X objective turret hole to the center turret position. The 63X turret hole is the only hole with a small Wollaston prism underneath it that can be manually adjusted with a thumbscrew. YOU MUST USE THIS HOLE FOR THE 63X. (Hole #1). The 10X Objective should be in Hole #2 and the 20X Objective should be in Hole #6)

5. Uncap the turret hole (MINIMIZE THE AMOUNT OF TIME THAT ANY TURRET HOLE IS UNCOVERED AS DUST CAN GET INTO THE HOLE WHICH CAN PERMANENTLY APPEAR ON EVERYONE’S IMAGES IN THE FUTURE).

6. Perform steps 6-11 over the Styrofoam bucket.

7. Unscrew the objective case cap from the objective case – OVER THE STYROFOAM BUCKET. NOTE: Be careful as there is no guarantee that the previous user screwed the objective into the objective case cap.

8. Make sure the objective is securely screwed into the objective holder cap.

9. Using a COTTON TIP APPLICATOR (LIKE A Q-TIP) AND NOT KIM WIPES (WHICH CAN SCRATCH THE OBJECTIVE) to wipe the excessive oil from the objective so that it is not slippery.

10. Inspect the objective for damage. FOR THE CURRENT DEMO LENS, a small indentation has already been formed at the point where damage occurs. You must ensure that this indentation does not become worse. We will take images so that you will have a comparison point for this. Please inform Phil immediately if you see anything.

11. Unscrew the objective from the objective cap.

12. Align the base of the 63X objective with the microscope turret hole (Hole #1, which is marked “1” on the microscope turret)

13. Gently screw the objective into the turret. Be EXTREMELY CAREFUL not to cross-thread the objective and turret. This will result in irreparable damage of many thousands of dollars. IF IT IS NOT SCREWING IN, DO NOT FORCE IT….UNSCREW IT AND TRY TO SCREW IT BACK IN. IF IT CONTINUES TO NOT WORK, STOP YOUR WORK AND TALK TO PHIL ABOUT IT.

 

Using the objective:

1. Apply lens oil to the objective lens. More oil is better than less oil.

2. Place your glass-bottomed sample in a specimen holder. You must ensure that the specimen glass has been wiped clean of salt crystals using dH2O and a Kim wipe. Salt crystals can scratch the objective lens just like Kim wipes!

3. Very SLOWLY bring the objective into contact with the specimen.

4. Look through the specimen to watch when the lens oil surface tension is broken by the specimen.

5. Use only the FINE FOCUS knob while looking through the eyepieces to focus your specimen. DO NOT USE THE COARSE FOCUS KNOB ONCE THE OIL MAKES CONTACT WITH THE SPECIMEN.

6. If your experimental protocol requires that you use a 10X, 20X and 63X objective on the same specimen, gently lower the 63X objective and switch to either the 10X or the 20X, then slowly bring the lower magnification focus up to the specimen The 10X Objective should be in Hole #2 and the 20X Objective should be in Hole #6. During this process, your number one task must be to ensure the safety of the 63X objective AS THIS COSTS AROUND $3000 TO REPAIR EVERY TIME IT GETS DAMAGED!!!. Check the location of the 63X objective after every repositioning of the 10X or 20X objective to ensure it is not contacting the stage.

7. If you wish to use the 5X or the 40X objectives, you must remove the 63X objective entirely.

8. During the use of the 63X objective, the center turret position must be on the 63X, the 10X or the 20X (the immediate right and left of the 63X turret position).

9. DO NOT CHANGE THE POSITION OF THE 10X, 20X, OR 63X OBJECTIVES BEFORE TALKING TO PHIL. They will be numbered and labeled to correspond with a specific turret position. The 63X belongs in position #1, the 10X in position #2 and the 20X in position #6

 

 

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Objective removal: Objective removal is the reverse of insertion.

 

1. Slowly lower the objective and remove the specimen.

2. Perform steps 3-6 over the Styrofoam box.

3. Unscrew the objective and screw it securely into the lid of he objective case.

4. Recap the turret hole immediately.

5. Using a cotton tip applicator (NOT A KIM WIPE) to remove oil from the objective.

6. Place the objective into the objective case by screwing the objective case lid into the objective case.

7. Store the objective in the objective drawer in computer desk located in the main laboratory (not the imaging computer desk).