Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

This study was performed with permission of the State of Saxony and Saxony-Anhalt, Regierungspraesidium Dresden/Halle, according to Section 8 of the German Law for animal protection (24D-9168.11-1/2008-24).

1 Cell Isolation

1.1 Preparation of Femur and Tibia

1. Anesthetize the mouse using a 5% isoflurane concentration by vaporizing isoflurane in a closed bin. After the mouse has stopped moving for 3 sec, perform the cervical dislocation.
2. Disinfect the hind limbs with ethanol (96%). Remove skin and muscles and separate the bones with a sterile scalpel.
   1. Start with an inguinal skin incision, and extend the incision toward the medial malleolus and perform a circular dissection of the skin around the lower calf. Remove the skin of the legs completely by peeling it proximally.
   2. Detach the quadriceps muscle from the femur with a sharp scalpel, remove both the anterior peroneus and the gastrocnemius muscle groups and disarticulate the leg at the hip joint by locating and dissecting the ligaments.
   3. Start anteriorly and proceed around the joint by carefully rotating the leg and transecting the ligaments, thereby disarticulating the joint.
3. Wash femur and tibia with 96% ethanol for a minimum of 90 sec to guarantee aseptic cell preparation. Continue the washing process with sterile PBS to rinse off remaining ethanol.
   Note: All subsequent steps must be sterile to avoid contamination.

1.2 Harvesting of Bone Marrow

1. Cut the proximal and distal end of each bone with a pair of fine scissors to gain access to the femoral and tibial shafts. Be careful with this step, since these bones break very easily.
2. Flush the bones with warm medium (M199 + 10% fetal calf serum (FCS), + 1% Penicillin/Streptomycin). Use a sterile 28-G needle, a 1 ml syringe, and between 10-15 ml medium per bone for rinsing.
3. Hold the bone with a fine forceps and rinse one by one until it turns semi-translucent. Carefully apply pressure to the end of the syringe plunger in order to minimize cell stress.
4. Filter the bone marrow through a 70 µm nylon web and gather the filtrate. To extract the maximum quantity of bone marrow, rinse repeatedly from the distal and proximal ends.
5. Rinse the sieve with PBS and gather the filtrate. Carefully dislodge debris and cellular conglomerates by gentle stirring and pipetting.

1.3 Cultivation

1. Centrifuge the cell suspension at 250 x g for 10 min at RT. Discard the supernatant and resuspend the cells with approximately 25 ml of medium. Repeat once.
2. Resuspend the cells in 6 ml of medium after the second washing step. Mix 50 µl of the cell solution with 50 µl of trypan blue. Count the cells in a counting chamber under a light microscope. Calculate the number of cells in the solution using this formula:
   
3. Seed the cells on 6-well ultra-low-attachment surface plates to prevent permanent adhesion to the bottom of the plate. Use a concentration of 10⁶ cells per ml with up to 6 ml per well.
4. Supplement the suspension with 20 ng/ml M-CSF to promote cell differentiation.
5. Culture the cells for 5 days at 37 °C and 5% carbon dioxide and observe daily.

1.4 Harvesting of Cells

Note: These steps should be performed on ice. Proceed with either 1.4.1 or 1.4.2 accordingly.

1. At this point, harvest the entire culture, including differentiated macrophages that will adhere to the culture-plates, even if they are ultra-low-attachment surface plates.
   1. Harvest the entire culture by gentle pipetting and detaching with a solution of 4 °C PBS, 0.5% bovine serum albumin (BSA) and 2 mM EDTA. Repeat until the plates are clear of remaining adherent cells – confirm under a microscope if unsure.
2. Alternatively, discard adherent macrophages by harvesting the supernatant with the cells in suspension, which predominantly contain monocytes.
   1. Harvest the non-adherent cells with a solution of EDTA-free PBS and 0.5% BSA. Do not apply too much force in order to avoid dislodging mildly adherent macrophages.

1.5 FACS-Analysis (Optional)

Note: It is possible to deplete the cell suspension of CD117⁺ stem- and progenitor cells by the use of MACS. Use manufacturer protocols for this procedure.

1. Harvest the cells according to either steps 1.4.1 or 1.4.2.
2. Centrifuge the cells at 250 x g for 10 min at 4 °C and repeat this step again.
3. Resuspend at least 250,000 cells in 300 µl of FACS buffer per probe.
4. Transfer cell solution onto a 96-well-plate (30 µl per well).
5. Centrifuge the cell suspension at 250 x g for 5 min at 4 °C.
6. Remove the supernatant and add 25 µl of antibody to each well (use manufacturer dilution).
7. Stain the cells with antibodies (following manufacturer protocols for cell phenotyping after steps 1.5.1-1.5.6. Commonly used markers for monocyte / macrophage identification include CD11b, F4/80 (Figure 3), CD115 (Figure 2), and Gr-1.
   Note: For a further description of cell solutions, the use of markers like CD4, CD8a, CD11c, CD25, CD45, CD45R, CD62L, CD80, CD86, CD145, CD117, MHCⅡ, Ly6C, Ly6G, and CD192 is recommended. Cellular characteristics were previously described¹².
8. Incubate the probes for 20 min at 4 °C.
9. Centrifuge the probes at 250 x g for 5 min at 4 °C and resuspend with 200 µl of FACS buffer. Repeat this step twice.
10. Transfer the probes into FACS tubes and perform the FACS analysis¹².
11. Perform FACS analysis¹² on day 0, 3 and 5 to analyze cell differentiation.

2. Tail Vein Injection

2.1 Preparation

1. Warm the animals on a heating plate at 37 °C for about 10 min. Observe the animals while warming to recognize signs of overheating.
2. Resuspend the monocytes, isolated in steps 1.1- 1.4, in 150 µl of NaCl, and load the cells into a 1 ml insulin syringe with a 30G needle. Lightly vortex the solution before loading the syringe to ensure all monocytes are injected. Always handle cells on ice to avoid heat-mediated attachment and activation of monocytes.

2.2 Restraining

1. Avoid disturbing other mice in the cage when choosing a mouse for intravenous injection.
2. Place the mouse into the restrainer. Avoid too much pressure and be careful with the hind limbs. (Figure 4)
   Note: Alternatively, use general anesthesia to guarantee stress free handling of the animals.
3. Ensure that the mouse has space for breathing before closing the restrainer.

2.3 Injection

1. Disinfect the injection site with disinfection agent. Spray on the tail of the mouse and let stand for at least 1 min.
2. Stop venous blood flow of the tail by applying gentle pressure to the lateral tail side above the injection site, for better visibility of the veins.
3. Turn the tail 90 degrees, before injection. (Figure 5)
4. Inject at a 45 degree angle. Inject slowly and no more than 5 µl per g to avoid harming animals.
5. If there is a blister, which is a sign of a failed injection, discontinue immediately and repeat the injection more proximally.
6. Stop the bleeding at the injection side by applying gentle pressure for about 1 min.
7. At the end of the procedure, open the retractor and place the mouse in its cage.
8. Observe the mouse for 20 min to ensure that the animal was not harmed by the injection and sternal recumbency is maintained. Extend the time of observation until the mouse regains sufficient consciousness. Return the mouse to the company of other animals only after it has fully recovered.