THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY

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1 Page: 1 of 5 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED Measurement of Glutamate Release using the FluoSTAR Optima Fluorescent Platereader. HAZARDS 1. Chemical exposure Magnesium chloride, NADP and Tris are all classed as Irritants - EGTA is classed as a flammable. 2. Animal bites or infection. 3. Cuts from guillotine RISK ASSESSMENT 1. Under standard laboratory conditions these chemicals pose a low risk, however suitable precautions must be taken to minimise exposure. 2. Low risk, laboratory animals are docile if handled gently and considerately. 3. Low risk. WRITTEN BY NAME (signed) Lynn Herd DATE August 2003 CHECKED BY Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 5 RISK CONTROL 1. Wear laboratory coat, enclosed shoes, gloves and safety glasses when handling any of the above chemicals. All chemicals must be appropriately labelled with Hazard warnings, Risk and Safety phrases. 2. Undertake an Animal Handling course. Only handle one animal at a time. If more than one animal is to be sacrificed, treat each animal as a separate experiment and clean up thoroughly between animals. Wear gloves, surgical mask and lab coat when handling animals. 3. Ensure guillotine is in good working order. Take care to remove fingers from area before operation of the guillotine. 2. Working Solutions: 2.1. Krebs Buffer Calcium free 23.6 ml 1M NaCl (118 mm final conc) 1.0 ml 1M KCl (5 mm final conc) g NaHCO 3 (25 nm final conc) 200 :l 1M MgCl 2 (1 mm final conc) g Glucose (10 mm final conc) Make up to 200 ml with dd H 2 O and bubble through Carbogen for 30 minutes. Keep on ice. Resume Carbogen bubbling 5 minutes before start of experiment and continue whilst reagent in use. **** Irritant - Wear Suitable Protective Clothing **** mm NADP g NADP Dissolve in 353 :l dd H 2 O. Make fresh daily. **** Irritant - Wear Suitable Protective Clothing, safety glasses and gloves, avoid contact with skin and eyes.**** mm CaCl g Calcium chloride FW = Make up to a final volume of 100 ml with dd H 2 O.

3 Page: 3 of mM EGTA g Ethylene Glycol Tetra-acetic acid FW = Add approximately 80 ml dd H 2 O and mix with a stirring bar. EGTA will not dissolve until the ph is adjusted. Using 5M NaOH adjust ph to 7.5. Make up to a final volume of 100 ml with dd H 2 O. **** Hazardous Flammable powder **** 2.5. L-Glutamic dehydrogenase solution (Sigma G mg) Solution in 50% glycerol, Na phosphate buffer, ph = mg protein per ml. 40 units per mg protein. One unit will reduce 1.0 :M ketoglutarate to L-glutamate per minute at ph 7.3 and 25ΕC in the presence of Ammonium ions mM L-Glutamate STOCK (1000x) g L-Glutamic acid FW = Make up to a final volume of 10 ml with dd H 2 O. (0.05 mmoles L-Glutamate in 10 ml; 5 nmoles L-Glutamate in 1 Φl) Φmole L-Glutamate (40x) Dilute 40 Φl 5mM L-Glutamate STOCK with 960 Φl Krebs buffer to give 0.2mM (40x). (0.2 Φmoles L-Glutamate in 1 ml; 0.2 nmoles L-Glutamate in 1Φl) add 5Φl. 3. Reagents: M NaCl g Sodium chloride FW = Make up to 1 litre with dd H 2 O. Store at 4ΕC M KCl g Potassium chloride FW = Make up to 200 ml with dd H 2 O. Store at 4ΕC M MgCl g Magnesium chloride FW = Make up to 50 mls with dd H 2 O. Store at 4ΕC. **** Irritant - Wear Suitable Protective Clothing **** 4. Procedure: 4.1. Prior to Test Prepare synaptosomes using GLP 025 Preparation of Percoll gradients and GLP 014 Preparation of Synaptosomes.

4 Page: 4 of Lowry Protein Estimation Resuspend Synaptosomes in 2ml ice cold Ca free Krebs buffer Perform Lowry Protein analysis on resuspended Synaptosomes. Dilute samples to1/4 and 1/10 for analysis. Standard curve 0 1 mg/ml. 60 :l sample volume Optimal protein concentration appears to be :g protein per well. This equates to an initial protein concentration of 2 3 mg/ml Prepare BMG FluoSTAR Optima Microplate Reader Turn on the FluoSTAR Optima fluorescent plate reader and the dedicated computer Log on and open the Fluostar programme The assay protocol GLUTAMATE_PLATE has been defined on the FluoSTAR Optima s a time-resolved Fluorescence (ie. kinetic) method in plate mode. It can be set to run for any length of time desired, optimising the number of cycles vs the number of samples assayed at any one time. It has been set up with the following parameters: Kinetic windows: 1 No. of cycles: ~80 Cycle time (s): ~15 sec Meas. start time (s): 0.0 No. of flashes: 10 Gain: 2500 varies with conditions Excitation filter: Emission filter: 460 Injection cycle 60 Volume group 1 Injection cycle 60 Volume group 2 Shaking after each cycle 7mm 1 second Turn on the temperature control to 37ΕC Prime the injectors with Krebs and 40x L-Glutamate depending on protocol to be followed A minimum of 1 ml of any solution is required for accurate operation of the injectors. 1.5 ml is recommended. Injectors may operate in Inverse Dispense mode to conserve reagent, and residual reagent may be recovered at end of experiment from tubing Setup the plate parameters:

5 Page: 5 of Setup microplate Samples are routinely set up in duplicate. An optimal number of samples to run appears to be 12. This allows 6 +Ca and 6 Ca-free conditions. There appears to be an artefact problem (jump in fluorescence) each time an addition is made, whether by injection or by stopping run and in injecting externally. To take this into account, a separate sample (X13) is run at the end of the experiment, and 5ul Krebs is injected into these wells at the same time as the 5ul Glutamate is injected into the other wells. The immediate rise in Fluorescence from the Krebs injection (the artefact) is subtracted from the Glutamate rise Resuspend protein as 2 or 3 mg/ml in Ca free Krebs buffer, using Lowry protein results. A full plate of experiments (96 wells) can usually be obtained from the sacrifice of one rat Add 25 Φl protein to each well Add 175 Φl Ca free Krebs to each well. (0 seconds) At 60 seconds add 2 Φl 100 nm NADP (or 5Φl 40 nm NADP) Immediately add 2.4 Φl 100 mm CaCl 2 to wells which will be +Calcium or 2.4 Φl 100 mm EGTA to wells which will be Calcium free At ~400 seconds add 2.3 Φl Glutamic dehydrogenase to each well Place microplate into FluoSTAR and begin recording fluorescence intensity If adding a drug add at 650 seconds Wait at least 200 seconds before adding 45mM KCl After 450 seconds add 1 nmole L-Glutamate. Wait at least 200 seconds before stopping experiment

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