A 4 hydroxytamoxifen (4 OHT) Preparation & Injection - michelletytong/TRGProtocols GitHub Wiki

Materials & Supplies

  • 100% Ethanol

  • Cremophor (Sigma C5135-1KG)

  • 4-hydroxytamoxifen (Sigma H7904-5MG)

  • Sterile vials (1.8mL) or 2.0 mL cyrotubes

  • 1.0 mL Eppendorf

  • Aluminum foil

  • Pipettors

  • Pipette tips with filtered tips

  • Needles and syringe (for mouse I.P. injections)

  • Hot plate

  • Vortex

Steps

Drug mixing/stock (Any time open 4-OHT be under the fume hood)

  1. Mix up fresh 10% bleach solution in a spray bottle (will be effective for 24 hours) or use bleach wipes

  2. Gather supplies/materials and label vials

  3. Use a pre-ordered vial of 50 mg of 4-OHT (Sigma H-____). (4-OHT powder is stored in the fridge (desiccated, protected from light, 2-8C).)

  4. Pipette 2500 ul of 100% EtOH directly into drug vial.

  5. Vortex briefly, and incubate in heated incubator (60°C for ~15 min) with occasional vortexing. Cap the vial

  • Resulting solution: 50 mg/2500 µl EtOH = 0.02 mg/µl = 20 mg/ml
  • 20 mg/ml is the ‘official’ maximum solubility of 4OHT in ethanol (Sigma spec sheet).
  1. Once the 4-OHT is in solution, add 2500 ul of Cremophor; mix well.
  • Resulting solution: 10 mg/ml in EtOH/Cremophor; total volume 5 ml.
  • Check that this ‘fits’ in Sigma vial; may need to move to other vial. If so, use a vial with volume measurements so can top off the EtOH if it evaporated.
  • If there is evaporation, can top off to final volume (presumably it’s the EtOH that evaporated, but can top off with cremophor to make mice less drunk – test this).
  1. Aliquot and freeze at -20°C.
  • Check volume before aliquotting; if evaporation occurred, top off to 5 ml total volume with EtOH to ensure correct concentration. Vortex before aliquotting.
  • 500 ul aliquots are a good choice: 5 ml --> ten 500 ul aliquots (each 10 mg/ml).
  • (Put into 1.5 to 2 ml cryotubes so that 1:1 dilution with PBS, making 1.0 ml, still has plenty of room to vortex).

Injection (to get 50 mg/kg body weight of 4OHT)

  1. Thaw aliquots of drug at 4°C for several hours or overnight.
  2. Weigh animals to determine appropriate volume of drug to inject.
  3. Thaw drug/EtOH/cremophor aliquot at 4°C for several hours or overnight
  • Dilute 1:1 with PBS; vortex. (so, 5 mg/ml final drug concentration, now in EtOH/cremophor/PBS emulsion.)
  1. (50 mg / 1000 g) x ( mouse weight in g ) x (1 ml/5 mg concentration) = ____ ml to inject
  • 20 g mouse --> 0.2 ml (for example)
  1. Load appropriate volumes of drug into syringes and keep at 37°C. Protect from light with foil.

Injection notes:

  1. Line tabletop with surgery surface liners prior to injection.

  2. (Mice generally will be being conditioned/exposed/etc. immediately before injection, depending on the experimental protocol in use).

  3. Inject animals I.P.

  • Note: all experimental animals should be acclimated to scruffing handling techniques weeks prior to the start of the experiment to minimize stress during injection.
  1. Return mice to their home cages after injection (since a new cage is novelty which is an activator) and to the mouse room. For Summer 2019, immediately after injection and return, expose mice to scented teaballs for 1 hour.

(5. Keep animals in their home cage for 5 hours (the time period of 4OHT activity). Make sure the animals have sufficient water. Use red lighting or turn off the lights during the wait period. Place a sign on the door if necessary to prevent accidental light exposures.)

Cleanup and notifications:

  1. Clean tabletops and other surfaces with fresh 10% bleach solution, followed by water (i.e. stainless steel table tops).
  2. Place all chemotherapy (tamoxifen-related) waste in yellow waste bags, the used syringes should go in the yellow sharps container.
  3. Make sure mice have abundant food in their cage lids since the cage will not be opened for 1 week.
  4. Label chemotherapy signs; notify animal care or other experimenters that no cage change required; hazard; special care for 1 week. Depends on the protocol for that institution.
  5. Place tamoxifen signs on cages in the animal housing room and on experimental room door.