Thiogycollate

Macrophage Recruitment with Thiogycollate

Purpose
In this experiment, we will stimulate macrophage recruitment in mice by injecting a thioglycollate solution into the intraperitoneal cavity. We will collect the macrophages produced by the mouse 4 days after injection and use this to culture the cells from each mouse. The macrophages obtained rom this experiment should be used within 1-3 days after harvest.

Materials

  • Thioglycolate solution (4% Thioglycolate)
    • Obtain Brewer Thioglycollate Medium (DIFCO Cat. No. 211716, 500g/each)
    • Reconstitute at 40.5g in 1 L of distilled or deionized water: Add 4.05g in 100mL ddH2O. Boil and stir until completely dissolved. Autoclave 2 days previous day of use. Store at 15-30°C; pH 7.2 ± 0.2 at 25°C.
  • Sterile troughs/Reagent reservoir (Corning Costar Incorporated Cat. No. 4871)
  • 3mL sterile syringes - 1 per mouse
  • Needles 27 gauge - 1 per mouse
  • Isolflurane anesthesia
  • 70% Ethanol for sterilizing
  • Mice: Record all information on mice injected (Cage, Mouse, ID, Sex, DOB, Genotype)

Protocol

  1. Fill syringes with thioglycollate (Do this inside the hood. Sterile.)
    1. Open thioglycollate bottle, inside the tissue culture hood only(autoclaved). Pour some thioglycollate into a clean, sterile trough.
    2. Insert needle into syringe.
    3. Uncap needle, making sure not to anything to it, as this part will touch the liquid. Needle and thioglycollate must remain sterile.
    4. Fill syringe to a level of 2.00ml, by carefully dipping the needle into the thioglycollate in the trough. Fill syringe until you reach level.
    5. Holding syringe straight up, tap the side of syringe to make any bubbles go to center top. Expel a small amount of liquid to get rid or bubbles.
    6. Cap syringe.
  2. Inject mice
    1. Uncap needle by holding down syringe horizontally, and lightly uncapping needle. Don’t take cap completely out. Leave this ready on the clean bench. Make sure there are no bubbles in syringe.
    2. Obtain mouse from cage.
    3. Anesthetize with a small amount of isoflurane...you don’t want to kill the mouse, do not leave mouse too long in the anesthetizing chamber.
    4. Take mouse out of chamber.
    5. Restrain mouse by gently, but firmly, pulling skin in the back of the neck. Hold skin between your thumb and the lower part of your index finger. Hold body of mouse in the palm of your hand, and pull the tail between and behind your pinky finger. On left hand.
    6. Obtain syringe with right hand, releasing cap. Inject syringe horizontally, directly into the intraperitoneal cavity (inject in lower
      abdomen). Insert entire needle.
    7. Inject thioglycollate solution gently, using your thumb to push needle pump.
    8. Inject until you reach 0.5ml level, so you inject 1.5ml of 4% Thioglycollate.
    9. Hold mouse firmly. If the mouse wakes up and moves, and you misinject, you will kill the mouse.
    10. Make sure mouse is ok. Put back in the cage.
    11. Mice: Record all information on mice injected (Cage, Mouse, ID, Sex, Genotype, Injected?)
  3. Stimulation for 4 days
    1. Leave mice for another 4 days to allow macrophage stimulation by thioglycollate in intraperitoneal cavity.
    2. Do not leave mice for longer than four days, or you will start getting lymphocytes as well.
    3. Do not leave for less than four days, or you will not get as many macrophages.
    4. Collect macrophages on the day after fourth day.