Isolation of thioglycolate-elicited peritoneal cells by peritoneal lavage

Materials:

Instrument tray Set of instruments (Forceps/scissors)

Trash bag Ice bucket/ice

70% EtOH squeeze bottle 10 ml syringes containing collecting medium

Hemostats 50-ml conical tubes (4)

Tube tray Small transfer pipets

1 ml syringe 3 ml syringes

Paper towels Latex gloves

Respirator Tape

Lab coat Marker and pen

Sleep Away (pentobarbital sodium) in 1 ml syringe

3% thioglycolate broth (Sigma) (autoclaved)

Collecting medium: 10 U/ml heparin/5% FBS in RPMI complete-18 ml/animal (keep ON ICE)

Cell wash medium: 5% FBS in PBS (keep ON ICE)

ACK-lysing buffer: 0.15 M NH₄Cl, 1 mM KHCO₃ and 0.1mM Na₂ EDTA, pH 7.2-7.4

RPMI complete

RPMI 1640 HEPES (Gibco) + 10% FBS (Atlanta Biol.)

100 U/ml penicillin

100 mg/ml streptomycin

2 mM L-glutamine

Protocol:

1. Inject 2ml of 3% thioglycolate solution into the peritoneal space of the animal 3 days prior to the lavage procedure.

2. 3 days after thioglycolate injection, euthanized the animals injecting 150-200 mg/kg of Sleepaway into the hind limb (intramuscularly).

3. After the heartbeat disappears (determined by palpating over the rib cage), take the animal and lay it on its back on 2-3 layers of paper towels.

4. Apply 70% EtOH to the chest/abdominal region.

5. Take the first set of sterile instruments and remove the skin from the ventral surface from the inguinal region to the trachea.

6. Inject 6ml of collecting medium into the peritoneal cavity with a 10 ml syringe and massage the abdomen.

7. Make a circular incision into the peritoneal layer on the mid-line (about 1cm in diameter)

8. Insert a transfer pipette through the hole in the peritoneum and clamp the peritoneal surface using a hemostat to form a tight seal.

9. Extract the peritoneal wash fluid and place it into a 50ml tube. Keep on ice.

10. Repeat injection of medium, massage and collection twice using fresh collecting media.

11. Centrifuge the tubes for 10 min at 1260 rpm at 4°C and discard the supernatant.

12. Wash the cells in 10 ml wash medium, centrifuge for 10 min at 1260 rpm at 4°C

13. Add 10 ml of ACK lysing buffer to remove the contaminating erythrocytes. Repeat if necessary to eliminate all red blood cells.

14. Wash the cells twice in 10ml wash medium, centrifuge for 10 min at 1260 rpm at 4°C.

15. Re-suspend the pellet in 1ml of complete media and determine cell count and viabilityby trypan blue exclusion.

16. Adjust volume for the desired cell/ml concentration and plate.

Modified by D.Bonilla from the guinea pig protocol (Laboratory Manual), according to Yamamoto T, 2007 and Saiki I, 1982.

References

Yamamoto, T. et al. Mycobacterium bovis BCG vaccination modulates TNF-alpha production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs. Tuberculosis 87, 155-65 (2007).

Saiki, I. et al. Adjuvant activity of purified peptidoglycan of Listeria monocytogenes in mice and guinea pigs. Infect Immun. Oct;38(1):58-65 (1982).