Faccio Lab

Roberta Faccio

Roberta Faccio, Ph.D

Professor

Department of Orthopaedics Washington University

faccior@wustl.edu
BJC - Institute of Health
11th floor - RM 11615
Phone: (314) 747-4602 

Protocols

Isolation of bone marrow macrophages (BMMs):
-Dissect long bones on your bench and place them into clean plates on ice
-Flush the bone marrow with alphaMEM (w/o serum) under the hood using syringes with 25g needle (usually 20ml for 1 mouse)
-Pipette well and pass cell suspension into a cell strainer (to get rid of little pieces of bones) (BD Falcon 70um)
-Spin down for 5min at 1000rpm
-Resuspend cells in alphaMEM + 10% FCS + 100ng/ml M-CSF and plate 5X106 cells per p100 petri dish (Fisher brand 08-757-12) or plate bone marrow from 1 mouse into 2 p150 petri dishes (Falcon 35-1058).
Cells will adhere in 2-3 days (these are BMMs)
-Lift BMMs with trypsin/EDTA (Invitrogen 15400-054 use 1:10), spin and resuspend in alpha MEM +10% FCS and use the cells to make osteoclasts or to expand BMMs.

Osteoclastogenesis:
-Resuspend BMM at a concentration 500’000 cells/ml.
-Prepare a 96 wells plate with 200 µl/well alphaMEM +10% FCS + 10ng/ml M-CSF and 100ng/ml GST-RANKL.
-Add 10 µl/well of cells (5000 cells/well).
-Change media every other day. Cells will start to round up by day2-3, and fuse to form multinucleated OCs by day4-5.
NB. FCS can be an issue. If you cannot make OCs check different lot# of serum!!

Bone resorption:
Use cow bone slices sterilized in EtOH for at least 1h and under UV light for 30 min. Wash them with sterile PBS and leave the bone slices at least 30 minutes in warm alpha-MEM.
Dispose bones in a 48 well/plate with 1 ml alpha MEM + 10 ng/ml M-CSF + 100 ng/ml RANKL. Add 25’000 BMM/well.
Change media every other day. After you saw the first OCs around the edge of the bone, wait additional 2-3 days, then recover bones and proceed with the Pit assay.
Detach cells from the bones using 2N NaOH and a cotton brush.
Wash the bone slices with PBS and incubate with 20 µg/ml peroxidase-conjugate wheat germ agglutinin (Sigma) for 30 min. Wash with PBS and add 3,3’-diaminobenzidine (0.52 mg/ml in PBS containing 0.1% H2O2) for 30 minutes. Bone pits can be evaluated as darker areas under light microscope.

BMM infection:
Day0 - Trypsinize Plat-e cells e plate them in tissue culture plates In the morning, at the density of 5 millions/plate in High Glucose D-MEM + 10% FBS. If cell density is not enough, change media in the evening.
- Sacrifice a mouse, collect cells from tibias and femurs and plate total bone marrow cells in p100 Petri dishes in alpha-mem +10%FBS + 100 ng/ml M-CSF at a density of 10 millions/plate.
Day1 - Transfect Plat-e cells in the morning (follow Trasfectol protocol).
D2 – Discharge first viral supernatant and replace media to Plat-e cells with fresh alpha-mem +10%FBS.
D3 - Collect and filter all viral supernatant from transfected PLAT-E cells. Use fresh filtered viral supernatant to transduce BMMs (10 ml supernatant /p100). Add M-CSF100 ng/ml M-CSF and 4 ug/ml of polybrene in each plate.
Leave in the incubator 24 hours.
D4 - Change media to transduced BMMs with new alpha-mem + 10%FBS and M-CSF. Add 1 ug/ml Blasticidin or 3 ug/ml puromicin (it depends on the vector).
D6 - Change media with new alpha-mem +10%FBS + 100 ng/ml M-CSF.
D7 - Transduced BMM are now ready to use! (OCgenesis, Western Blot or Bone resorption assay…).

Left ventricle (LV or intra-arterial) bone metastasis model and In vivo bioluminescence imaging and analysis:
For intra-arterial injections, operators were blinded to genotype. Mice were anesthetized and inoculated intra-arterially with 105 B16-FL cells in 50 ml PBS as previously described. Tumor growth was monitored by in vivo bioluminescence imaging on Days 8, 10 and 12 following B16-FL tumor injections. Bioluminescence imaging was performed using a charge coupled device (CCD) camera (IVIS 100, Xenogen, Alameda, CA) as described. Mice were shaved to minimize attenuation of light by pigmented hair. On the days indicated, mice were injected intraperitoneally with 150 mg/kg of D-luciferin (Biosynth, Naperville, IL) in PBS ten minutes prior to imaging. Mice were then anesthetized using isoflurane and imaged (exposure time one or five minutes, binning 8, FOV 15cm, f/stop 1, no filter). For analysis, total photon flux (photons/sec) was measured from a fixed region-of-interest (ROI) in the bones or whole body using Living Image 2.50 and IgorPro software (Wavemetrics, Portland, OR).

mBSA Induced Arthritis:
D0: Immunize the mice by injecting s.c. at the base of the tail 100 µl of mBSA (final concentration 1 µg/µl) in Complete Freund Adjuvant and 100 µl of pertuxin toxin IP.
D7: Repeat immunization in the same way as day 0 (use Incomplete Freund Adjuvant).
D21: anesthetize mice and inject 10µl [10µg/µl] mBSA into the left knee and 10 µl PBS into the right knee.
D30: sacrifice the mice and take:
       - bones for histology: leave bones at RT for 24 hours in Formalin, then 14 days in 14% EDTA, then perform different washing with 30%, 50% and 70% EtOH (1 hour for each wash).
inguinal Lymph nodes for TH1/TH2 test: recover Lns in RPMI no serum, smash them, wash and count cells. Plate Ln cells in 96 well plate with 200 µl RPMI + 10% mouse serum, + mBSA in vitro (50 µg/ml). After 3 days, recover supernant, and analyze cytokine production using TH1/TH2 test assay from BD bioscience.