The wild type C57BL/6 mice (age 8-12weeks) obtained from the Jackson laboratory. The mice housed per the guidelines of Xuzhou Medical University, Jiangsu Province, China. The use committee approved the animal studies of Xuzhou Medical University (Xuzhou, China) and the National Institute of Health guide for the care and use of laboratory animals, Jiangsu province, China. (Acceptance number: XZMC20130226).
Step-by step process of bone marrow extraction:
Mice aged 8–12 weeks terminated by cervical dislocation, and the whole mice soaked in 70% ethanol for 2-5minutes, then placed on the sterile dissecting board on the laminar flow hook. The long bones of the femora and tibias pulled off with micro-dissecting scissors placed in sterile Dulbecco's phosphate buffer solution (DPBS).
The muscles were detached from the bones by forceps, and the bones scrubbed to remove any residual soft tissues. The bones were washed twice with DPBS solution containing 1mmEDTA. One edge of the long bones cut –off place in a new dish containing the DPBS/EDTA solution.
An 18G needle pushed to the bottom of the 0.5 ml nest microcentrifuge tube. The cut edge of the long bone inverted downwards in the 0.5 ml nest microcentrifuge (maximum of 2 tibias and 2 femora) and the lid closed.
The nest of the 0.5 ml micro-centrifuge tube transfer into a 1.5 ml centrifuge tube, sealed with Parafilm and centrifuge at 15,000 g for 30 seconds. The nest 0.5 ml microcentrifuge discarded and the visible pellet at the bottom of the 1.5 ml Eppendorf tubes suspended with sterile DPBS/EDTA solution.
The suspended bone marrow cells with the PBS/EDTA solution, filtered with 70um cell strainer (Biologix group limited) and centrifuge at 300 g for 5 minutes at room temperature. The pellet resuspended in DMEM supplemented with 20% fetal bovine serum (FBS), and 500 ul of penicillin (10,000uints/ml) /streptomycin 10,000ug/ml.
The cells plated in a sterile 10 mm dish with a minimum of 109 cells/plate and incubated in a 5% CO2 humidified incubator overnight.
Step-by-step Process of isolating bone marrow-derived endothelial cells:
After overnight incubation, the floating cells poured off. The adhered cells were washed with pre-warmed DPBS twice and detached with appropriate accutase solution for 15 minutes at room temperature.
The detachment stopped by adding the harvesting buffer PBS, 2% FBS, 1 mm Ethylenediaminetetraacetic acid (EDTA), and penicillin/streptomycin) with repeated pipetting to detached the remaining cells. The detach cells transferred into 15 ml tubes, centrifuge at 300 g for 10 minutes at room temperature. The total cell number determined by hemocytometer or automated cell counting machine.
The pellet cells resuspended with the harvesting buffer solution CD31 microbeads by manufacturer's protocols and stored at 40 C for 15 minutes.
The cells resuspended with 10 ml of harvesting buffer solution, then centrifuge at 300 g for 10 minutes at room temperature to wash off the excess beads from the cells after 15 minutes incubation.
The MS column attached to the magnetic washed once with the harvesting buffer, bone marrow cells pass through the column. The magnetic cells were washed three times with the harvesting buffer followed by a single wash with endothelial cell medium containing the factors.
The magnetic cells pushed into new sterile tubes with the endothelial cell media containing the additional growth factors, the total cells count determined by hemocytometer or automated cell counting machine.
The plate pre-coated with rat-tail collagen type 1, wash with sterile DPBS solution twice followed one wash with the endothelial cells growth media. Appropriate cell number seeded into the coated dishes and incubate into the incubator.
CHARACTERIZATION OF PRIMARY BONE MARROW-DERIVED ENDOTHELIAL CELLS (BMDECS).
Bone endothelial cell structure visualization
The cultured cells sequentially observed every day for the capillary-like structure appearance of endothelial cells with confocal microscopy and photographed. (Nikon Eclipse Ti microscopy, Tokyo, Japan).
Matrigel capillary tube formation assay of BMDECs
Matrigel was allowed to thaw on ice overnight according to the manufacture's protocols. Prechilled 24 well plates were coated on ice with 200 ul Matrigel per well. Gels were incubated for 30 minutes for solidification at 37 0 C. BMDECs 10^5 cells, resuspended in 500 ul of the pre-cold endothelial cell medium supplemented with growth factors and cell plated on the gels. The plated cells were incubated in the humidifier at 370 C for 4–7 days. The morphological changes are periodically monitored and photographed.
Identification of bone marrow endothelial cells by flow cytometry analysis
The selected CD31 positive cells analyzed by flow cytometry with a panel of antibodies; [CD31 (PE. anti-mouse, eBioscience™), CD45 (FITC anti-mouse, eBioscience™) CD106 (PE, Rat-anti-mouse, BD-Pharmingen™), CD144 (APC, anti-mouse, eBioscience™) and ESAM (APC, anti-mouse, Biolegend®) antibodies]. 500,000cells/ml were collected per tube, washed with PBS, centrifuge at 400 g for 5 minutes at room temperature (x2), incubated with the recommended dilution of antibodies, store at 40 C for one hour, and analyzed by flow cytometry (BD LSRFortessa ™) within 24 hours. The lower threshold uses to exclude debris and the live cells with gating (20,000 cells) according to FSC x SSC, followed by sections containing the antibodies. The data retrieve from the flow cytometry software and analyze by flow Jo software version 7.6.2.
Characterization of primary bone marrow-derived endothelial cells by Real-time quantitative PCR (RT-qPCR)
To verify the molecular expression of the bone marrow endothelial cells, total RNA extracted from the cells after 7 days of incubation and the negative cells immediately after isolation using Trizol reagents (TIANGEN Cat#dp424). The cDNA is synthesized by using 5X All in one RT Master mix (Cat.No.G492) and kept at -20 0C. Primers sequences and probe are shown in (Table 1). For RT-qPCR, the synthesized cDNA samples 10 ng were amplified with the SYBR green master mix in a final volume of 20 ul, as described in our previously published article(13). The mean threshold values are used to evaluate the molecular gene expression with normalization with mouse beta-actin.
Validation of bone marrow endothelial cells by immunoblotting analysis
To examine the molecular expression of bone marrow endothelial cells. The cells were placed in a plate pre-coated with rat tail collagen type 1 washed with cold PBS, and a herpes-chap lysis buffer containing the protease inhibitors pour into the dish, incubate for 10 minutes, and adherent cells scraped off with cell scraper. The lysed cells were centrifuged at 20,000 g for 30 minutes at 40 C. While For the non-endothelial cells (CD31 negative), the protein isolated immediately after isolation and protein stored at -200C until ready for use. The cell supernatants run on SDS-PAGE 8–12% gel (BIO-RAD; Hercules, CA). The proteins were transferred to the P 0.45 PVDF blotting membrane (Amersham™Hybond™ Germany) by the wet transfer method. Primary and secondary antibodies are shown in (Table 2).
Characterization of BMDECs by immunofluorescence staining
To certify the bone marrow endothelial cells for the expression of CD31 (PECAM-1), VE-cadherin (CD144), and ICAM-1 with passage zero determine by direct immunofluorescence staining. The CD31 positive cells were plated into pre-coated 48-well with rat tail collagen type 1 for 5–7 days, as described above. After 60–70% of the confluence, the medium removed, cells fixed with 4% paraformaldehyde (PF for 20 minutes at RT, followed by two wash 3 minutes apart with PBS. The cells were permeabilized with 100% cold methanol at room temperature for 20 minutes, rinse with PBS three times and blocked with 1% BSA/PBS for 1 hour at room temperature. Cells incubated with the recommended dilution of primary antibodies overnight at 40C. The cells were cleaned twice with PBS and counterstained with Hoechst for 5 minutes at 370C. The cells were washed with PBS and imaging acquired using inverted Nikon microscopy (Nikon Eclipse Ti microscopy, Tokyo, Japan). Primary and secondary antibodies are shown in (Table 2).
INDUCTION OF PRIMARY BONE MARROW DERIVED ENDOTHELIAL CELLS BY RECOMBINANT TUMOR NECROSIS FACTOR ALPHA (TNF-α).
Assessment of primary bone marrow-derived endothelial cell proliferation
To examine the cell proliferation of primary bone marrow-derived endothelial cells, 105cells were seed in 48 well plates incubated for 7 days. After 7 days, the BMDECs was stimulating with recombinant TNF-α (10 ng/ml) and the control with 1% FBS with DPBS for 48hrs, the media containing the recombinant cytokine and FBS/DPBS were replaced with new endothelial media and incubated for another 7 days. The cells were harvest by trypsin/ETDA ( VICMED 0.25% 0.02%), centrifuge at 350 g for 5 minutes, wash 2x with PBS, and fixed with 70% cold –ethanol at -200C for 1hour. The fixed cells centrifuge as above followed by washed with FACS buffer incubated with Ki-67 (FITC, anti-mouse, Biolegend®) and F4/80 (APC, anti-mouse as isotype control Biolegend®) for 30 minutes at room temperature. The data acquired by flow cytometry. For the cell count, the cells were stain with trypan blue and live cells counted by hemocytometer.
The initiation of bone marrow endothelial cells by recombinant tumor necrosis factor-alpha
The molecular expression of markers specific for bone marrow endothelial cells verifies by; real-time quantitative polymerase chain reaction (RT-qPCR) or immunoblotting and immunofluorescence staining. The cells were cultured for 7 days to form a confluence, then stimulated with TNF-A at (10 ng/ml) and vehicle control (2% fetal bovine serum in PBS) for 48hrs. Cells were then harvested for RT-qPCR, western blotting analysis and stained for immunofluorescence, all samples done in triplicate, and results expressed in mean with standard deviation.
All data were statistically analyzed using Graph Prism 6 and paired two-tailed student's test used for comparison mean ± standard deviation. P-values ≤ 0.05 are considered statistically significant.