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- 10mM EF5 in normal saline
- Prepare four 50 ml sterile pp tubes, 0.22µ millex-gs filter, 10 or 20 ml syringe. EF5 drug should be stored at room temperature in dessicator.
- Fill on 50 ml tube with filtered water and mark it as such.
- Tare second tube in a suitable container (lightweight pp 3-cournered beaker) on the micro balance.
- Weigh our tissue culture grade NaCl at 9mg/ml (0.009g/ml). Aim for enough for about 45mls (0.405g). Note that you do not aim for an exact amount of salt; you adjust the amount of added water. Thus for 420 mg you would add 420/9 or 46.7ml of water. Add this directly to the tared tube, assuming a density of 1.00.
- Remove tube from balance, add cap and shake to dissolve salt.
- Weigh third 50ml tube and record; tare to zero.
- Add EF5 to tube, at 3mg/ml and aiming for ~35ml; so 105 mg. As with salt, you adjust the amount of liquid, not the drug, but in this case the premixed saline is added, not water. e.g. suppose you had 103mg of EF5 (0.103g). Saline to add is 103/3 or 34.3ml → add 34.3g of saline. Add cap tightly.
- Tip at 37° to dissolve (sonnication may be necessary to break up any flakes or larger drug pieces).
- Remove the plunger from the syringe barrel; attach filter with gentle twisting motion, keeping outflow side sterile (**do not screw on too tight as this will force all the solution through a very tiny portion of the filtration membrane**).
- Add the EF5-saline solution to the syringe barrel.
- Insert barrel while aiming output into final sterile tube and filter entire solution. When you have a larger volume then can fit into one syringe, save the sterile cover for the filter. At the end of each filtration (don't add more than the specified amount, even though more could be fit into the syringe barrel) remove the filter and place it sterile end down into the cover, remove syringe barrel, reinsert syringe into filter, add more solution and continue filtering by adding back barrel and depressing.