XENOGRAFT: Imagingtumors expressing luciferase using xenogen equipment

Protocol, 5/18/2016

Cell Injection/Luciferase Imaging Sedation Protocol For Mice

(Mouse injection and imaging are performed in HCI LL155)

Prepare cells for injection

  1. use luciferase-expressing cell lines for injections.
  2. trypsinize cells and suspend in media + appropriate antibiotic.
  3. mix 50ul + 50 μL trypan blue and count cells on hemocytometer.
  4. calculate total cell number.
  5. pellet cells 1500rpm/5min and immediately remove media.
  6. re-suspend pelleted cells in appropriate volume of sterile 1×PBS to obtain 1×107 cells/mL (1x106 cells per 100 μL injection). Make dilutions as needed. ***Keep on ice***

Injecting the cells into the mice---1x Injection for each experiment

NB: VERY IMPORTANT to keep the cells well mixed in between injections

  1. Thoroughly mix cells, break up any clumps that there may be, by drawing up and down into a 1 mL syringe with a cannula attachment. Alternately, draw directly up via 27-gauge needle, but in this case draw slowly so you don’t destroy the cells. If you draw up enough cells to inject all the mice, make sure to keep the cells well-mixed between animals (try inverting, and/or flicking the syringe).
  2. Draw up cells into the same 1 mL syringe and place 27-gauge needle onto it.
  3. Tap syringe with needle pointing upward and push out all air bubbles. The following will be for subcutaneous injections rather than orthotopic injections.
  4. Grab the mouse near the tip of its tail (mouse should be facing away from you).
  5. Wrap your pinky finger at the base of its tail using your most distal knuckle. Keep the tip of the tail between your index finger and thumb.
  6. Grab the scruff with your thumb and index finger while still holding onto the tip of the tail. Can use your middle finger to keep the back straight or arched.
  7. One you have a firm hold on the mouse then place the needle just under the skin(subcutaneous) in the right or left dorsal flanks injecting 100 μL of cells.Note- you should see a small fluid filled region develop. Ensure that the needle is subcutaneous, and not too deep. When the needle is subcutaneous, you can almost see the needle through the skin. The plunger should depress easily.
  8. Repeat step 6-7 for the contralateral flank (2 total injections per mouse)

Luciferase Imaging Injections of mice-24 hr following cell injections, then, 1x per week

  1. Luciferin injection mix should be @ 20oC. Injection aliquots (D-Luc)—4 mL aliquots without Avertin—enough for 5-6 injections —Thaw @ RT covered in foil.
  2. OR—make up, in t.c.hood, one 4ml aliquot per 5 mice to be injected
  3. D-Luciferin @ 20oC (7.5 mg/mL)2668μL
  4. Avertin @ 20oC (1000mg/mL) 48μL

[dissolve 1g Avertin in 0.63 mL isoamyl alcohol]

  1. Sterile H20 884μL
  2. Sterile 10× PBS 400 μL
  1. In mouse Procedure Room LL155, add in 48μLAvertin (1000 mg/mL) per 2 mL aliquot---supplies are in the locked cabinet[key in cup, locker 22] and this should be done in the hood in room. Mix aliquots by inverting a few times.
  2. Plug in heating pad, from locked cabinet, in the hood and set to high.Place mouse cages on the heating pad (keeps mice from going hypothermic after sedative is injected).
  3. Set up the Xenogen IVIS system for imaging according to the mouse imaging-Xenogen IVIS Protocol.
  4. Draw up the sedative into a 1mL syringe with 27-gauge needle (can use the same needle for as many injections as you can fit into the syringe).
  5. Tap syringe, with needle pointed upward, and push out all air bubbles.
  6. Note:::::Make note of what time you conduct your first mouse and last mouse injections for a given set of mice and time 5min following the final injection before imaging them and I advise only doing one cage at a time as the mice do not stay sedated for anymore than 20-30min.
  7. Grab mouse near the tip of its tail and either:
  8. allow mouse to grip the rails of its cage upside down
  9. OR, hold the mouse upside down just inside the cage gently restraining its headagainst the cage wall with gentle pressure just behind its head in the neck area.
  10. Stick the needle into the area between its abdomen and leg with a slight upwardpush until the needle is mostly inside of the animal (the animal is upside down at this point and you should be pushing the needle in the direction of the tail).
  11. Inject 0.75 mL of sedative per 30g mouse for the final concentrations per mouse of the following (i.e. 20g mouse =0.5mL or 500μL):
  12. D-Luciferin50 mg/kg
  13. Avertin300 mg/kg
  14. Following the final injection set a timer for 5min then image mice according to the mouse imaging-Xenogen IVIS Protocol
  15. Place mice back in their cage when finished and allow them to wake up whilestill on the heating pad (I usually wait until they are all at least waddling around before putting them back in their usual room LL125).

Preparation of D-Luciferin stock:

  1. D-luciferin: Xenogen (cat # XR-1001; 1 g vial, ~$850)
  2. Make 75 mg/mL stock:
  3. dissolve 1 g D-luciferin (use whole vial) in 13.1 mL sterile dH2O, filter through 0.22 μm syringe filter.
  4. store this at -20°C to dilute later (wrap in aluminum foil)
  5. Make 7.5 mg/mLworking stock:
  6. 1 ml of the 75 mg/mL stock D-luciferin + 9 mL sterile dH2O
  7. can freeze at -20°C (wrap in aluminum foil)
  8. NB: each 10 mL stock makes about 7.5 of the 2 mL injection stocks.