Overview
This video demonstrates a peritoneal lavage method for isolating mature connective tissue-type mast cells from the peritoneal cavity of adult mice. Upon isolating a mixture of primary immune cells from the peritoneal cavity, selective proliferation of mast cells is performed under appropriate culture conditions.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. PMC isolation by intraperitoneal lavage
- Prior to the cell isolation, prepare the materials listed in Table 1.
- Use 8 to 14-week-old male mice for PMC isolation. Euthanize a mouse by CO2 inhalation, and confirm the death by loss of reflexes. Spray the mouse with 70% ethanol and fix it on a foam block using pins (dorsal side down). Remove the ventral skin of the mouse using blunt edge scissors. Avoid damaging the peritoneal cavity.
NOTE: We use typically this protocol for mice with a C57BL/6N strain genetic background. - Inject 7 mL of the ice-cold RPMI medium and 5 ml of air in the peritoneal cavity using a 10 mL syringe equipped with a 27 G needle. Push the needle carefully in the peritoneum and do not perforate any organs. Use a spot in the region of the epididymal fat to reduce the risk of organ perforation.
- After injection, shake the mouse for 1 min to detach peritoneal cells into the RPMI medium. Do not shake the mouse too strongly, to avoid damaging the internal organs and contaminating the peritoneal cavity with the blood.
- Reuse the 10-mL syringe by equipping it with a new 20 G cannula. Shift the inner organs to one side by tilting the foam block and gently tapping it on the bench to make medium aspiration easier from the other side. Insert a 20 G needle, bevel up, and aspirate the fluid from the abdomen gently and slowly (~0.5 mL/s) to avoid clogging by the inner organs. Collect as much fluid as possible (typically 5–6 mL).
- Remove the needle from the syringe and transfer the collected cell suspension to a collection tube on ice. Discard a sample tube if there is visible blood contamination.
- Centrifuge the tubes with the cell suspension at ~300 x g for 5 min. Under a sterile hood aspirate the supernatant. For each mouse, combine the sample pellets in 4 mL of cold (4–10 °C) PMC Medium and transfer the cell suspension to a 25 cm2 culture flask. Add the growth factors IL-3 and SCF to the final concentrations of 10 ng/mL and 30 ng/mL, respectively. Place the flask containing the cells in an incubator (37 °C and 5% CO2) and incubate for ~48 h until the next procedure.
Table 1: Materials for Step 1.
| 1 | Ice |
| 2 | 10 mL syringes |
| 3 | 27 G needles |
| 4 | 20 G needles |
| 5 | Styrofoam block and pins |
| 6 | Collection tubes (50 mL Plastic Centrifuge Tubes) |
| 7 | 70% ethanol |
| 8 | RPMI Medium (Pre chilled and kept on ice) |
| 9 | PMC Medium: RPMI Medium + 20% FCS (Fetal Calf Serum) + 1% Penicillin Streptomycin solution (Pen-Strep) |
| 10 | Dulbecco’s phosphate buffered saline - DPBS (without Ca2+ and Mg2+) |
| 11 | Culture Flasks (25 cm) |
| 12 | Growth factors stock solutions (IL-3: 1 μg/μL; SCF: 2.5 μg/mL) |
| 13 | Serological pipettes (10 mL) |
| 14 | Pipettes tips sterile (20–200 µL) |
| 15 | Hemocytometer |
| 16 | Bench Centrifuge |
| 17 | Scissors and forceps |
| 18 | CO2 chamber for mice |
| 19 | Open sterile hood |
| 20 | Closed sterile hood |
| 21 | Cell incubator (37 °C and 5% CO2) |
| 22 | Transfer pipettes (20–200 µL) |
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Materials
| Name | Company | Catalog Number | Comments |
| RPMI Medium | Thermo Scientific | 21875034 | |
| DPBS | Sigma-Aldrich | 14190144 | |
| FCS | Thermo Scientific | R92157 | |
| IL-3 | R&D Systems | 403-ML | |
| SCF | Thermo Scientific | PMC2115 | |
| Penstrep | Thermo Scientific | 15140122 | |
| Culture Flasks (25 cm) | Greiner Bio-One | 69160 | |
| 15 mL Plastic Centrifuge Tubes | Sarstedt | 6,25,54,502 | |
| Bench Centrifuge | Heraeus | Megafuge 1.0R |
