MATERIALS

• 10ml sterile syringes (one for each mouse)
• 20 gauge needles (one for each mouse)
• Phosphate Buffered Saline, pH 7.2: PBS 1X (GibcoBRL Cat. No. 20012-027)
• Isoflurane for anesthetizing
• 70 Ethanol for spraying
• Instrumentation, autoclaved: 2 surgical scissors, forceps, curled fine forceps
• 50ml conical tubes, sterile (one for each mouse genotype)
• Macrophage media: 20 FBS in DMEM high-glucose + penicillin:

-         50ml Fetal Bovine Serum (ATCC Cat. No. 30-2020), thaw at 37° C before adding (20FBS)

-         2.5ml Penicillin-Streptomycin (Invitrogen 15140-122), thaw at 37° C before adding (1)

-         197.5ml DMEM high-glucose, +L-glu, no Na-pyruvate (GibcoBRL Cat. No. 11965-092). Thaw at 37° C before adding

-         Combine all and filter. Store at 4° C.

Note: Can use FBS or Pen-Strep from any company, as long as it has been kept sterile and you use it consistently throughout the experiment.

• Ice bucket
• 6-well plates for tissue culture
• Mice:
• All procedures will be performed in the tissue culture hoods, following tissue culture preventive procedures
• Mice injected with 1.5ml of 4 Thioglycollate 4 days prior:

PROTOCOL

Macrophage Extraction

Obtain all materials. Autoclave instruments. Prepare media as specified and keep at 37° C. Obtain mice. Fill 10ml syringes with sterile PBS 1X and cap with 20 gauge needles. Mark each with mouse number, genotype and identification. Place on ice. Note : Aliquot PBS into a 50ml falcon tube when filling syringes, so there is less change of contamination of entire PBS bottle. Fill syringes from 50ml falcon tube. Anesthetize mouse in anesthetizing chamber

-         Add a small amount of isoflurane to tissues in bottom of chamber.

-         Place mouse in chamber.

-         Watch for breathing of mouse and take out when it slows down. If mouse dies from anesthesia in this step, it will not be a problem.

Euthanize mouse by cervical dislocation

- Hold mouse by nape of neck firmly and pull tail with other hand until you feel a pop and a separation between cranium and backbone.

5. Sterilize mouse

- Place mouse in a beaker with 70 ethanol.

- Place mouse in tissue culture hood immediately.

6. Laporotomy incision

- Make a small incision in abdominal skin of mouse (use one set of forceps and scissors).

- Open up mouse by pulling skin with both hands to each side of mouse. Do this firmly so that skin is completely pulled down, but gently so that you do not open up intraperitoneal cavity or break any limbs. Spray peritoneum with 70 Ethanol.

7. Inject Cold PBS

-  Uncap needle inside the hood and squirt out a small amount of PBS to get rid of any bubbles.

- Inject needle, hole-side down, into the mouse in lower abdominal area, preferably in a region where there is fat.

- Insert needle gently and not too deeply so you do not perforate any organs (it is important not to get blood in the IP cavity).

- Immediately after inserting the needle, begin injecting PBS into IP cavity forcefully.

- Take needle out slowly so that you allow fat to clog the hole (if you take it out too fast, the PBS will come out of the hole).

8. Wash mouse

- Hold mouse by tail and swish around to wash (lavage) IP cavity with the PBS that has been injected.

9. Macrophage extraction

- Using same syringe, go to upper part of abdomen and insert needle horizontally.

- Make sure you do not perforate any organs.

- Important: Hold needle inside peritoneal cavity in a horizontal position, with the needle hole-side down, and extract the PBS containing macrophages. Do this slowly; if you do this too fast, you will aspirate fat and clog needle.

- Try to get at least 7ml of fluid.

- If you needle, insert again in another part, again lifting a tent and extracting the fluid.

- Cap needle again and place back on ice. Make sure it has proper genotype of mouse.

* Repeat this procedure for each mouse.

Plating

Collect cells

-         Pour cell suspension in 50ml collection tubes.

-         You may pool suspensions of different mice with same genotype.

-         Centrifuge cells at 1Krpm for 10 min. Make sure you balance the centrifuge.

-         Discard supernatant: Pour off supernatant inside tissue culture hood so you keep only pellet. Discard into another sterile tube, in case you loose pellet while pouring off.

ACK Red Blood Lysis (Optional)

- Re-suspend pellet in 10ml ACK Lysis Buffer and incubate on ice for 15 minutes.

- Pellet again under same conditions (1k for 10 min).

- Discard supernatant (pellet should be clean), and resuspend pellet in 10ml PBS.

- Pellet again and discard supernatant.

Note: Do this only if you see a lot of blood in pellet. The least you handle the cells, the better.

Re-suspend cells

-         Using 10ml pipette, add Macrophage media (previously prepared) to the pellet: 3ml per mouse.

-         Re-suspend cells by pipetting up and down. At first, do so close to the cell pellet, releasing fluid horizontally over the walls of the tube so as not to lyse the cells. Later, keeping doing this but higher over the cell pellet. This must be done relative quickly and firmly so as no suspend cells properly, but not too forcefully so that you do not lyse the cells. Also, do not release fluid all the way down so that you don’t get bubbles. Be careful not to aspirate all the way up or you will touch filter and contaminate the cells. Watch the pipette and do this very carefully , several times, until pellet is complete dissolved.

Plating Counts: Determining cell concentration in macrophage cell suspensions

Prepare dilutions

-         Add 20μ l of cell suspension to 80μ l of cold PBS (5X dilution).

-         Take 10μl of this­ 5x dilution + 10μl of Trypan blue dye (2X dilution).

-         Use 10μ l of this 10X dilution and pipette into counting chamber.

Counting

-         Count number of cells in each square of the chamber.

-         Average the number for all 4 squares in chamber.

-         Use this number to determine [Macrophages]:

[Macrophages] =     # Average of cells x 10 (DF) x 1x104 = _____________cells/ml

Dilute cell suspension

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