© copyright, all rights reserved / ISOLATION AND CULTURING OF MOUSE BONE MARROW DERIVED MACROPHAGES / Code:
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Author 1 / Anouk Hamers / PhD student / July 2009 / AAJ
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1Title

Isolation and culturing of mouse bone marrow derived macrophages (BMMF)

2PRINCIPLE

Mononuclear phagocyte progenitor cells derived from femoral and tibial bone marrow are propagated in the presence of M-CSF. This macrophage growth factor is secreted by L929 cells and is used in the form of L929 cell conditioned medium. Within 8 days the progenitor cells proliferate and differentiate through monoblast, promonocyte and monocyte stages before maturing to macrophages. At this time the cells become firmly adherent to the culture vessel. Other cell types present will not attach to the culture vessel.

The bone marrow is obtained by flushing the femurs and tibia with ice cold sterile PBS.

3AREA OF APPLICATION / LIMITATIONS

Department:Medical Biochemistry.

Type of material: hindlegs from mice, fresh or stored in PBS at 4°C.

Only people who are registered article 9, 12 and 14, described in the Dutch law ‘Wet op Dierproeven’, are allowed to work with animals. When not registered as such, you are only allowed to start at paragraph 8.2 of this protocol.

4DEFINITIONS AND TERMS

BMMF: Bone Marrow Derived Macrophage

Pen/Strep: Penicillin/Streptomycin

FCS: Foetal Calf Serum

5SAFETY

  • Work according to the general laboratory safety protocols as described in the most recentversion of the Laboratory Safety Manual.
  • Wear gloves to prevent skin contact.
  • When bitten by an animal clean your wound with disinfectant. If you haven’t been vaccinated for tetanus within the last 10 years, then go straight to the emergency room. If you are vaccinated for tetanus and the wound is not big, cleaning the wound should be enough. If the bleeding doesn’t stop,then go to the emergency room.
  • More information about laboratory safety can be found at the AMC intranet: Laboratory safety course (Laboratorium Veiligheidscursus):

6REAGENT, STANDARDS AND CONTROLS

6.1Chemicals

Chemicals / Manufacturer / Product no. / Molecular weight/
Molarity / R code(s) / S code(s)
Ethanol 96% / Orphi Farma / 174837 / 46.07 g/mol / 11 / 7-16
PBS / Fresenius Kabi Nederland B.V. / M090001/01NL
FCS / Invitrogen / 10270-106
Penicillin/
Streptomycin / Invitrogen / 15140-122
Lidocaine hydrochloride / Sigma-Aldrich / L5647-15G / 270.8 g/mol / 22 / 36
RPMI 1640 / Invitrogen / 52400-25

Ethanol: 96% 5L containers, order via the labassistant.

PBS: sterile solution of PBS: Fresenius Kabi GmbH, REF: M090001/01NL, store at RT.

FCS (=Foetal Calf Serum): Invitrogen, REF: 10270-106, batch no.: 41F5675K (see also remarks), -20ºC. See SOP General cell culture techniques (Anita).

LCM (=L929 Conditioned Medium): See SOP Production of L929 Conditioned Medium. Store at -20ºC.

Medium RPMI 1640(+HEPES + l-glutamin):Invitrogen, REF: 52400-25, store at 4ºC.

Penicillin/Streptomycin: 10,000U/ml Penicillin,10,000μg/ml Streptomycin. Invitrogen, REF: 15140-122, -20ºC, AMC-nr: 1030978. See SOP General cell culture techniques (Anita).

Lidocainehydrochloride: Sigma-Aldrich, CAS73-78-9, REF: L5647-15G, batch no.:034K0573, store at RT.

6.2Solutions

Bone marrow macrophage medium (freshly made before each isolation):

RPMI with HEPES

Penicillin/streptomycin

10% FCS

15% LCM

For each mouse you will need up to 100ml.

Now the medium can be kept at 4oC for 1 week.

Note: If the tube of LCM is thawed, do not freeze it again.

Lidocaine solution:

Transfer approximately 200mg oflidocaine powder to a 50ml tube.

Add up to 50ml of warm sterile PBS (you will need 12.5ml for each culture plate)

Sterilize the solution through filtering using a 0.2µm filter placed on a sterile 50ml syringe.

7MATERIALS AND EQUIPMENT

Materials / Manufacturer / Product no.
2 petri dishes p/s 145/20mm bacterial quality / Greiner / 639161
2 Syringes (50ml) / BD Plastipak / 300865
A 21 gauge needle (0.8x50mm) green / BD Microlance / 301155
4Tubes 50mlpolypropylene / Greiner bio-one / 227 261
1 Tube 15mlpolypropylene / Greiner / 188271
1 Culture flask (200ml/80cm2) / Nunclon / 153732
0.2µm Filter / Whatman / 10462200
A 25 gauge needle (0.5x16mm) orange / BD Microlance / 300600
Equipment / Manufacturer and type
Small scissors / Medicon
Large scissors / Medicon
Dissecting forceps / Medicon, 07.56.39
Centrifuge / Hettich Rotanta/P
Waterbath (37oC) / Memmert
Incubator (37oC, 5% CO2) / Boom B.V., NAPCO CO2 5400
Cell counting chamber (0.0025mm2, 0.04mm2) / Marienfeld, Bürker
Kleenex tissues

8PROCEDURE

8.1General preparation

  • Sacrifice the assigned mouse by means of CO2 asphyxiation (70% O2 and 30% CO2 for 1min, then 100%CO2for 4min).
  • Disinfectthe hind legs of the mouse using 70% ethanol.
  • Cut the hindlegs free from the rest of the body using large scissors without breaking the bones (femurs and tibia).Keep the leg straight and the femur head intact!
  • Remove/strip the skin away from the hind legs and rinse them with 70% ethanol.
  • Transfer the legs to a 14ml tube and put them on ice.
  • Move to a culture hood at the department of Medical Biochemistry (K1-165).
  • Put one 50ml tube of sterile PBS on ice.
  • Prepare the BMMF medium (§6.2)

8.2Analysis

Bone marrow isolation:

  • Transfer the legs to the culture hood (put them on tissue paper) and clean them with 70% ethanol.
  • Cut the achilles tendon and remove the paw using scissors. When removing the paw, make sure you cut behind the ankle joint (press the scissors against the calcaneum and cut).
  • Remove the flesh from the legs as far as possible using small scissors (don’t cut into the bones!).
  • Cut the fibula from the tibia.
  • Remove the remaining flesh using a tissue drained with 70% ethanol. It is important to make sure that all the tissue is removed from the bones, since cells associated with this can contaminate the marrow preparation and potentially overgrow the macrophages.
  • Cut off the patella.
  • Separate the tibia from the femur at the knee joint. (Bend tibia in opposite direction from natural bending of the knee).
  • Again, clean femurs and tibia with 70% ethanol.
  • Fill a 50ml syringe with ice cold sterile PBS up to 20ml and attach a 25 gauge needle (orange). The PBS has to be ice cold because otherwise the macrophages will attach to the tube.
  • Cut off both ends from the bones (just beneath the heads).
  • Hold the bones firmly above a 50ml tube using tweezers and flush the bone marrow with the ice cold PBS (use 20ml per mouse). Flush on both sides of the bones! Keep the flushed bone marrow on ice! (or else the macrophages will attach to the tube)
  • Centrifuge the cells for 5min at 1200rpm.
  • Aspirate the PBS and resuspend the pellet in BMMF medium by aspirating and flushing twice using a 10ml syringe with a 21 gauge needle.
  • When flushing for the second time seed the cells onto two 15cm bacteriumplastic culture plates (5ml/plate) and make sure it is spread well (macrophages tend to attach very quickly after seeding).
  • Add 20ml BMMF medium to each culture plate.

Culture the cells for 8 days at 37oC and 5% CO2.Add 15ml of fresh BMMF medium on the 4th day of culture. After 8 days the macrophages can be harvested to use in experiments.

Harvesting BMMF:

  • Prepare a lidocaine solution (§6.2).
  • Aspirate the BMMF medium from the culture plate and add 12.5ml of lidocaine solution.
  • Incubate for 10min at 37oC.
  • Resuspend the lidocaine in the plate until all BMMF have detached. When the BMMF are still attached to the culture plate it appears blurry. After the BMMF have detached from the culture plate it looks clear.
  • Transfer the lidocaine solution containing the BMMF to a 50ml tube and centrifuge for 5min at 1200rpm. Keep the BMMF on ice!
  • Aspirate the lidocaine solution and resuspend the BMMF well in 10ml cold BMMF medium.
  • Count the cells using a Bürker counting chamber.See SOP Counting cells using a Bürker counting chamber (Mariska).
  • Bring the concentration at 8x105cells/ml.
  • Now the BMMF can be used in experiments, e.g. 1ml for a 12-wells plate and 500µl for a 24-wells plate.

9QUALITY CONTROL

Not applicable.

10RESULT PROCESSING

Not applicable.

11ACCURACY AND PRECISION

Not applicable.

12REMARKS

The amounts of substance/solutions, materials and equipment used are applicable to the bone marrow isolation and subsequent BMMF culture from one mouse.

The functionality of FCS is batch-dependent and should be tested before ordering a new batch.

13DOCUMENTS APPLICABLE

SOP Countingcells using a Bürker counting chamber

SOP Production of L929 Conditioned Medium

SOP Working in culture room

SOP General cell culture techniques (Anita)

14FORMS APPLICABLE

Not applicable.

15REFERENCES

Not applicable.

16APPENDIXES

Not applicable.

Department of Medical Biochemistry, AMC, Amsterdam, The Netherlands