Metzger Lab Protocol Book Written by Pierre Coutu 08/06/2003. Ionoptix Procedure

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1 1. Overview: Ionoptix Procedure The Ionoptix system allows the user to measure simultaneously unloaded sarcomere shortening and calcium fluorescence in isolated cardiac myocytes. The cells have to be plated on 18mm x 18mm glass cover slips at a density of 20,000 myocytes per cover slip (see heart prep. procedure). The experiments are generally (but not necessarily) performed in modified media M199 and fluorescence is measured with calcium sensitive dye Fura-2AM. Important note: The system can be used to measure sarcomere shortening only. All the steps involving calcium fluorescence measurements will be typed in italic. 2. Material: 2.1 Equipment: List: Ionoptix Station: Grass stimulator, Electronic Thermometer, Nikkon Microscope, data acquisition computer, Ionoptix components (Xenon Arc Power Supply, Stepper Switch, Fluorescence System Interface, and Video Power unit) 2.2 Supplies: List: 2.3 Reagents Qty Item 2 50mL conical tubes 1 15ml conical tubes 1 6-well plate Several glass pipettes Several 18mm x 18mm coverslips List: a) Modified M199 (M199+): - One bottle of Sigma M199 (Sigma-#M2520) g sodium bicarbonate g glutathione g Bovine Serum Albumin (BSA) - Adjust volume to ~950ml with distilled water - Adjust ph to 7.4 (using NaOH)

2 - Adjust volume to 1L with distilled water in a volumetric flask - Add 10ml of P/S - Filter under the hood into sterile bottle (suggestion: 2x 500mL Strericup filter system). b) 1mM Stock Fura2-AM: - Add 49.9uL of DMSO to a tube of Fura2-AM (for final [] 1mM) - Shake (10s) and spin (10s), keep at 20 C. Good for 2 weeks. 3. Methods: 3.1 Equipment preparation and setting: Temperature control: If experiments are to be performed at temperature other than room temperature, turn the water bath on ½ hour before the start of the experiment. (Note: Dial in temperature control should be set to ~40-41 C in order to have a temperature of 37 C in the experimental chamber). Make sure the water level in the bath is within ½ inch from the top. Warm up ~50ml of M199+ in a conical tube (you might need more or less depending on the type of experiments) Xenon Arc Lamp: If you are measuring calcium fluorescence, the first thing to be turned on is the xenon arc lamp power supply (to avoid electrical surcharge on the other equipments) Rest of the equipment: Turn the following pieces of equipment on: - Grass stimulator - Microscope lamp - The Ionoptix Stepper Switch box (top shelf/ 2 nd box) - The Ionoptix Video Power box (left side of the microscope) - The Ionoptix Fluorescence System Interface box (left side of the computer) - The electronic thermometer (left side of the microscope)

3 3.1.4 Settings: Instrument Control Value Comment Grass stimulator S1 repeat/s1 off/ S1 off (center pos.) Off until ready to S1 Repeat use SR1 Rate (PPS) 2.0 (dial) X 0.1 (knob) 0.2Hz or a pulse every 5 seconds S1 Delay Minimal values No delay S1 Duration 5.0 (dial) X 1.0 (knob) Pulse duration of 5ms S1 Voltage 3.0 to 4.0 (dial) X 10 (25Ω) (knob) ~30 40 Volts S1 Stimulus On (top position) Power is available Ionoptix video power controller 60/120/ (bottom pos.) 240 images / seconds Fluorescence System Interface Man/AGC Man (top position) Manual gain control PMT* On Off When meas. Fluo. Otherwise ON/OFF On Other toggle switches Bottom positions Knobs Leave as is * PMT should never be on when the ambient lights are on. It might damage the equipment! 3.2. Fura-2AM loading: Preparation: - Warm up ~13 ml of M199+ into a 15ml conical tube (37 C) - Pour 2.5ml of warm M199+ into one of the well of a 6 well plate and label : F-2 - Pour 3.5ml of warm M199+ into three chambers of the 6 well plate and label : W1, W2, W3. - Add 12.5uL of the 1mM Fura2-AM stock in well labeled F Loading: - Put the cover slip (with the myocytes plated on) in the F-2 well For rat: 4½-5 min incubation time at 37 C For mice: 4-4½ min incubation time at 37 C Note: The Fura2-AM/M199+ mixture can be used for up to 3-4 hours. After that, make new mixture (see step 3.2.1).

4 3.2.3 Washing: - At the end of the incubation period, put the cover slip into wells label W1, W2, W3 for 5-10 seconds each. Leave in W3 until ready to use. Note: Try to keep loading time as consistent as possible (at high [] Fura2 buffers calcium and attenuates shortening) 3.3. Mounting the cover slip on the microscope: - Add a drop of distilled water on the objective - Lower the objective - Add the cover slip to fully cover square opening on the stainless steel heating plate. - Screw the stimulus chamber on top, insert the temperature probe, and add M199+ to fully cover the stimulus chamber apparatus. - It is recommended, but not mandatory, to add an empty cover slip on top (to avoid leakage, evaporation, and vibrations) - Connect the two electrode leads to the stimulus chamber. Make sure that the Grass stimulator is on the S1 off position. Notes: i) Don t waste too much time while fixing the cover slip in position. Otherwise the cover slip will dry and the myocytes will die. ii) It might take up to 5 minutes to reach desired temperature at the beginning. Temperature varies during the experiment. Make sure to look at the electronic thermometer regularly during the experiment and adjust water bath temperature dial if necessary. iii) You can keep your solutions warm in the small incubator beside the Ionoptix station. 3.4 Finding cells: - Move the objective up, until you see the water drop entering in contact with the cover slip (watch from above the cover slip). - Put the Port View knob to Eye position and adjust focus - Press the single (red button) on the Grass stimulator to find contracting cells 3.5 Acquiring data: Directory Setup (to be done once): On the computer: - Select: My Computer C: - Select: File New Folder

5 - Give it your name (that s where you data will be stored) - You can subdivide your directory as you want (one folder per experimental session usually works well) Acquisition: - For calcium measurements, turn ambient lights off - Select the Acquire Icon and click OK (first time only) - Select: File New Sarcomere Length / PMT Acquisition - Select (First time only): Collect Parameters o In the Experiment Name window Select: User (for Sarcomere Length only exp.) Select: Calcium (for S.L. and Calcium Fluo exp.) - Set the microscope Port View knob to Side position - Adjust the camera position such that the cell lies entirely in the visible field and is in a horizontal position (striations should be vertical). - Place the yellow window on a portion of the cell that has nice striation pattern and optimize to have the best possible sarcomere length pattern. o The black line represents light intensity vs position (should be sinusoidal) o The green lines are the upper and lower sarcomere length for which the program is looking (values are display on the right side : values between 2.0 and 1.4 should be OK) o The red line is the Fourier transform of the black line as function of sarcomere length. It should have a single and definite peak between the green lines. - Turn the Grass stimulator to S1 Repeat position and wait for 5-10 beats - Select: Collect Start and wait until the end of the sweep. - Turn the Grass stimulator back to S1 off position - Select: File Close - It should ask you if you want to save the file, click yes and save the data into your own directory. - Find another cell and repeat Notes: o For calcium fluorescence measurements the lights have to be turned off. o For calcium fluorescence measurements, it is recommended to first select an area where there are no cells, and proceed with the acquisition. This data becomes the background (the background will have to be removed during data analysis). One background measurement at the beginning of each cover slip should be sufficient. o For all measurements, it is recommended to limit the number of cells measured per cover slip to ~12-16 (or 1 hour). Media should be replaced after each block of 8 cells (if paced only during measurements) or for each 3-4 cells (if paced continuously). You can temporarily dispose of old media in a 50mL conical tube.

6 3.6 Cleaning up: - Close all equipment (the xenon arc lamp last) - Dispose of everything you used that was not there before you started. o Bleach used cover slips (and M199 wastes if the myocytes were in contact with virus) o Dispose of used glassware in glass waste and plastic in biohazard waste o Make sure you don t leave any solutions in the incubator - Clean stimulus chamber with 70% ethanol, hot water, and distilled water - Clean table, microscope, and stainless steel heating stage with 70% ethanol - Make sure there are no broken cover slips left on the table - Put back tweezers, screws, stimulus chamber on top shelf The station should be in good and clean conditions, and ready for the next user! 3.7 Transferring data: The acquisition station is for measurements. Your data should be transferred (using a Zip disk) to other computers for analysis. The analysis program should be installed on all the computers.

7 3.8 Analyzing data: Background: - Open the analysis program - Select: File Open (your background data) eg. cell0_bkg - On the fluorescence ratio curve, left-click and drag o Note the average values for denominator and numerator Data analysis: - Open the analysis program - Select: File Open (your data) eg. cell0 - Select: Operations Constants o In the Constant Types window select: dual numeric background o Enter the numerator and denominator values obtained in Select: Operations Average Events o Use the +/- selected, +/- hilite, >>, and << in the middle of the screen to remove undesired pulse from the average. - Select: Traces Show base traces o The check mark indicates that the option is active, by removing the check mark you are making it inactive. Now you should be left only with the average traces. - Click in the sarcomere length trace window (anywhere) - Select: Marks Add transients o Acquisition method: Convert from mark event o Event Mark text: Equivalent TTL mark o Analysis Range: Selected o Offset: anywhere from -.01 to -.1 (rat -.05) o Duration: depends on your traces: anywhere from 0.2 to 2.0s (rat 0.8) o Erase old marks: (should be active) And click OK - Select the Trans button o Make sure the thick color curves fit your data reasonably well and that there are no important ERROR messages in the values reported. You can play with the Offset and Duration parameters (see previous points) to obtain better results. - Select: Export Transient Analysis 3 Clipboard main (or simply use the shortcut: Ctrl-F) - Paste the result in an Excel spreadsheet

8 3.8.3 Spreadsheet organization: - It is recommended that you preserve an area for pasting in your worksheet. From there, copy and paste the data in an appropriate place for further statistical analysis. - In general, one Excel file per experiment works fine. - You will probably need one worksheet for SL data (plus another one for Calcium data) per cell group (e.g. control, lac-z, ctni, ) Parameters to analyze: Here s a list of typical parameters that are used in the analysis report. They can be selected or unselected with: Operations Transient Analysis Options - Baseline (bl): o Value of the signal just before the transient mark (stimulus) - Departure velocity (dep v): o Maximal speed obtained between the stimulus and the peak of your signal. (There are three sub-options. You don t need to select any of these) - Peak (peak): o Peak (minimum or maximum of your signal) information. The basic information (peak) indicates the absolute value. If you select the height suboption, it will calculate the difference between peak and baseline to give you the change in amplitude (peak h). You should also select the Include Time sub-option. It gives the time from stimulus to peak (peak t). - Return velocity (ret v): o Maximal speed obtained between the peak and the end of your signal. (There are three sub-options you don t need to select any of these). - Time to % Peak (or % Baseline) o You can select to report the time when the signal reaches a percentage of its amplitude. In general, for both the Peak (between stimulus and peak) and the Baseline (between peak and end), the 25%, 50%, and 75% are used. o You can either select the time value to be specified from t0 (stimulus) or from tpeak (time of peak). You can select one (e.g. t0) and calculate the other one in the spreadsheet (e.g. time from peak to 50% relaxation = time from_stimulus_to_50%_relaxation time peak) - Exponential Fit o Use mostly for calcium transient. The option fits from XX% return works well (90% is a good value). It fits an exponential curve {Offset+Amplitude*exp(-(t-t_start)/tau)} between XX% of the return and the end of the signal (position of the marker) - The other options are rarely used.

9 3.8.5 Filtering: - For calcium transient, signal-to-noise ratio might be small. You may want to use the filter option. - To select a filter configuration that works well for the calcium signal: o Select: Operation Raw filter setup Select: Savitzky-Golay Filter Select configure: width 11, Order 4 o Click OK-OK (to close the two windows) - To apply filter, select the filter button above the trace Note: Be careful, if you use a different filter configuration, make sure it you doesn t change the shape of your signal. For example the initial default setting (Low pass Butterwoth filter: 10Hz) will remove all the fast components of your signal and will therefore deform it.