Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells

Summary

The present protocol describes a cost-effective method to purify, expand, and characterize highly homogeneous bone marrow mesenchymal stem cells (BMSCs) from mice. This approach facilitates the acquisition of large quantities of BMSCs with high proliferation and differentiation potential, supporting mouse models and advancing preclinical research.

Abstract

Mesenchymal stem cells (MSCs) are a population of stem cells that can self-renew and differentiate into multiple cell lineages. Among them, MSCs found in bone marrow, known as bone marrow mesenchymal stem cells (BMSCs), play a crucial role in osteogenesis, hematopoietic support, homing/migration, and immunosuppression. BMSCs are widely used in cell biology and tissue engineering research due to their potential for cellular therapeutic strategies, as well as their easy availability, genetic stability, and low immunogenicity. Hematopoietic stem cells (HSCs) and BMSCs coexist in bone marrow but have distinct progenitor cells. When extracting cells from bone marrow, multiple cell types are typically obtained, which can lead to heterogeneity in the cell population, potentially causing bias and not meeting high experimental standards. Therefore, it is crucial to develop effective techniques to improve the homogeneity and yield of BMSCs in vitro. This study presents a detailed protocol for the purification, expansion, and flow cytometry-based phenotyping of BMSCs obtained from BALB/c mice. The homogeneity and stem cell phenotype of third-passage BMSCs were characterized using flow cytometry, demonstrating positivity for CD29, CD44, and Sca-1, negativity for CD45, and CD31, and confirming their osteogenic and adipogenic differentiation ability. This cost-effective protocol provides an easy and efficient method to obtain a sufficient number of homogeneous BMSCs with high proliferation and differentiation potential, which is essential for advancing research in mouse models and extensive preclinical studies.

Introduction

Bone marrow mesenchymal stem cells (BMSCs) are known for their multipotent characteristics and are widely used in the treatment of various diseases due to their self-renewable, differentiation, and immunomodulatory properties^1,2. BMSCs are readily obtainable, capable of extensive expansion, exhibit immunosuppressive properties, and do not trigger immediate immune reactions³, making them appealing for tissue engineering applications and effective carriers for cell therapy.

In the bone marrow, two main stem cell populations and their progenies, the hematopoietic stem cells and the BMSCs, are the primary residents⁴. Traditionally, BMSCs are obtained through targeted bone marrow aspiration and identified as plastic-adherent cells in vitro. However, this method is insufficient to differentiate BMSCs from other bone marrow subpopulations, including endothelial cells, pericytes, leukocytes, and hematopoietic stem cells⁵. Therefore, an easy and effective protocol for isolation of BMSCs is needed.

The current work details the separation and purification of BMSCs through their physical adherence to plastic cell culture plates. Flow cytometry experiments were conducted on third passage BMSCs using the surface markers CD29, CD44, and Sca-1, as outlined by the International Society for Cell & Gene Therapy (ISCT) Mesenchymal Stromal Cell (MSC) committee⁶. According to ISCT criteria, mouse MSCs are characterized by positivity for CD29, CD44, and stem cell antigen 1 (Sca-1), and negativity for hematopoietic cell marker CD45 and endothelial cell marker CD31. Additionally, BMSCs should be multipotent, demonstrating trilineage differentiation into osteoblasts, chondrocytes, and adipocytes. In this experiment, osteogenesis was induced in the extracted BMSCs, with ALP staining performed on day 7 and an alizarin red S assay on day 21 to assess differentiation. Adipogenic differentiation of isolated and purified BMSCs was also induced, with Oil Red O staining performed on day 21 to assess adipogenesis.

Protocol

Animal handling was approved in accordance with the Laboratory Animal Ethics guidelines (Ethical approval No.: PZSHUTCM2211070009) from Shanghai University of Traditional Chinese Medicine. Eight-week-old female BALB/c mice (SPF grade, 20 ± 1 g body weight) were used in this study. This protocol outlines the purification, passaging, flow cytometry-based phenotyping, and osteogenic and adipogenic differentiation of mouse-derived BMSCs. The reagents and equipment used are listed in the Table of Materials.

1. Animal handling

1. House mice under specific pathogen-free (SPF) conditions in individually ventilated cages, with five mice per cage. Provide SPF-grade feed and sterile water. Maintain a 12-h light/dark cycle.
   NOTE: This method has been tested and found to be feasible in both the BALB/c and C57BL/6 mouse strains. This paper mainly uses BALB/c as an example.

2. Harvesting femurs and tibias from mouse hind limbs

1. Euthanize the animals via CO₂ asphyxiation followed by cervical dislocation⁷ following ethical guidelines.
2. Soak mice in 75% ethanol for 5 min (Figure 1A).
   NOTE: Stem cells were harvested within 30 min² after euthanasia, and the entire process, including marrow flushing, filtration, and seeding into culture plates, was completed within 2 h to ensure optimal cell viability.
   NOTE: Use adult mice (≥8 weeks old) for optimal BMSC yield; 8–12 week old mice are ideal⁸.
3. Make an incision about 1 cm at the hind limb of the Achilles tendon with sterile dissection scissors (Figure 1B).
4. Make a circular incision around the hind limb of the Achilles tendon with sterile scissors to expose the distal tibia (Figure 1C).
5. Peel the skin from the incision towards the hip to remove it from both hind limbs (Figure 1D).
6. Cut along the iliac bone to separate the femoral head from the hip bone (Figure 2A).
   NOTE: Avoid damaging the bilateral joints of the femur during dissection to prevent the loss of bone marrow.
7. Place the collected femurs and tibias in a 60 mm dish harboring sterile pre-cold PBS containing 2% penicillin/streptomycin (Figure 2B).
8. Cut off as much tendon and muscle as possible from the tibia and fibula using a sterile scalpel to facilitate later flushing (Figure 2C).
9. Return the processed bones to PBS containing 2% penicillin/streptomycin and transfer them to the sterile hood for subsequent isolation steps.

3. Purification and culture of mouse BMSCs

1. Prepare complete MEM Alpha Modification (αMEM) medium for BMSC culture by supplementing αMEM with 10% Fetal bovine serum and 1% penicillin/streptomycin.
   NOTE: Choose αMEM medium since it contains 1000 mg/L of glucose, which is a relatively low concentration. This helps prevent the inhibition of cell proliferation that can occur in high-glucose environments⁹
2. Take a 50 mL syringe and attach the needle that comes with the syringe.
3. Draw 20 mL of αMEM medium into the 50 mL syringe, then replace the needle with the one that comes with the 1 mL syringe.
   NOTE: Use a 1 mL syringe needle to accommodate the bone size and minimize the cell damage. A 50 mL syringe allows for efficient flushing of the bone marrow with reduced risk of cell contamination from repeated insertions.
4. Use scissor to cut off both ends of the separated femur or tibia in a sterile hood.
5. Insert the needle into the bone along the cut edge and flush the bone marrow into a 100 mm culture dish containing complete αMEM medium (Figure 2D).
6. Flush bone cavities repeatedly until the bone pieces appear pale to ensure that most bone marrow has been extracted (Figure 2E).
7. Pipette up and down 40 times gently with a pipette tip to disperse the aggregated cells as much as possible.
8. Filter the cell suspension through a 40 μm cell strainer into a 50 mL centrifuge tube to remove debris, tissue fragments, and undispersed clumps.
9. Transfer the filtered cell suspension to a 100 mm cell culture dish and add complete αMEM medium to a final volume of 15 mL per dish.
   NOTE: Culture all the bone marrow cells collected from the femurs and tibias of each mouse in one 100 mm culture dish to facilitate cell growth. Based on previous experiments, approximately 1 × 10⁷ BMC can be isolated from an 8-week-old mouse (from two femurs and two tibias). These cells are seeded into a 100 mm culture dish at a density of 1.8 × 10⁵ cells/cm² for primary culture.
10. Hold the dish with both hands and gently swirl it in a figure-eight motion 15 times to ensure an even distribution of cells across the dish.
11. Place the culture dish at 37 °C in a 5% CO₂ incubator and culture for 5 days (Figure 3A).
    NOTE: Avoid moving the culture dish during this period to ensure uniform cell growth.

4. Replacement of αMEM medium for BMSC culture

1. Observe the cells under a microscope to determine if they have reached 80% confluency. If not, proceed with the medium replacement on the 5^th day.
2. Draw 3 mL of PBS with a sterile Pasteur pipette and dispense it from a height of 1 cm above the culture dish at different positions.
   NOTE: Avoid dispensing liquid down one side of the dish, as this may remove adherent cells due to excessive impact force and prevent effective removal of unwanted cells.
3. After adding 3 mL of PBS, gently shake the culture dish to thoroughly wash the cells, and remove PBS from the dish with a pipette. Repeat the wash process once more to ensure the cells are thoroughly washed.
4. Draw 15 mL of complete αMEM medium with a sterile Pasteur pipette and dispense it from 1 cm above the culture dish at different positions.
5. Return the culture dish to the incubator and monitor until cells reach 80% confluency before subculturing (Figure 3B).

5. Passage of BMSCs

1. Wash 80% of confluent cells twice with PBS (Figure 3C).
   NOTE: Washing the cells with PBS before digestion helps remove residual medium and cell secretions and reduces MSC adhesion to the culture dish.
2. Digest the cells with 1 mL of 0.25% trypsin-EDTA by incubating at 37 °C in a 5% CO₂ incubator for 2–3 min.
3. Neutralize the trypsin by adding 4 mL of complete αMEM medium to the cell suspension.
4. Aspirate the cell suspension from the culture dish using a 1 mL pipette tip, and gently flush the bottom of the dish to detach all adherent cells.
5. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 300 × g for 5 min at room temperature.
6. Use a pipette to discard the supernatant and resuspend the cell pellet in 2 mL of complete αMEM medium.
   NOTE: For subculturing, a seeding density of approximately 4 × 10⁴ cells/cm² was found to be optimal for continued growth and proliferation.
7. Split the cells at a 1:1–1:2 ratio into new 100 mm cell culture dishes (this constitutes the 1^st passage, P1) (Figure 3D).
8. Split the cells approximately every three days at a 1:1.5 ratio when they reach 80% confluency to generate the second passage (P2) (Figure 3E) and the third passage (P3) (Figure 3F) of BMSCs.

6. Preparing P3 BMSCs for phenotypic identification and osteogenic differentiation

1. Discard the cell culture medium.
2. Wash the P3 BMSCs with 3 mL of PBS  twice.
3. Digest the P3 BMSCs with 1 mL of 0.25% trypsin-EDTA by incubating at 37 °C in a 5% CO₂ incubator for 2–3 min when they reach 80% confluency.
4. Neutralize the trypsin by adding 4 mL of complete αMEM medium to the cell suspension.
5. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300 × g for 5 min at room temperature, and discard the supernatant.
   NOTE: The P3 BMSC pellets prepared here will be used for phenotypic identification via flow cytometry and for assessing osteogenic or adipogenic differentiation.

7. Phenotypic identification of BMSCs by flow cytometer

1. Resuspend the cell pellets of P3 BMSCs with 1.5 mL of PBS solution containing 2% FBS and adjust the concentration to 5 × 10⁶ cells/mL.
2. Aliquot 100 μL of the cell suspension into each of 11 tubes to achieve 5 × 10⁵ cells per tube. Prepare the following groups: label blank, CD29 isotype control, CD29, CD44 isotype control, CD44, Sca-1 isotype control, Sca-1, CD31 isotype control, CD31, CD45 isotype control, and CD45.
   NOTE: In the current study, the following fluorochromes were used: CD29-FITC, CD29 isotype-FITC, CD44-APC, CD44 isotype-APC, Sca-1-PECy7, Sca-1 isotype-PECy7, CD45-PerCPcy5.5, CD45 isotype-PerCPcy5.5, CD31-PE, and CD31 isotype-PE (as shown in the table of materials). These fluorochromes can be modified based on the configurations of different flow cytometers.
3. For each group, add the following reagents: 2 μL of PBS to the label blank group, 2 μL of CD29 isotype to the CD29 isotype control group (1:50 dilution), 2 μL of CD29 to the CD29 group (1:50 dilution), 0.5 μL of CD44 isotype to the CD44 isotype control group (1:200 dilution), 0.5 μL of CD44 to the CD44 group (1:200 dilution), 0.5 μL of Sca-1 isotype to the Sca-1 isotype control group (1:200 dilution), 0.5 μL of Sca-1 to the Sca-1 group (1:200 dilution), 2 μL of CD31 isotype to the CD31 isotype control group (1:50 dilution), 2 μL of CD31 to the CD31 group (1:50 dilution), 2 μL of CD45 isotype to the CD45 isotype control group (1:50 dilution), and 2 μL of CD45 to the CD45 group (1:50 dilution).
   NOTE: Antibodies were used according to the manufacturer’s optimized recommendations for flow cytometry. Preliminary experiments confirmed optimal staining and specificity for target cells. Refer to the manufacturer’s dosage guidelines and conduct titer pre-tests as needed.
4. Mix gently, centrifuge using a palm centrifuge, and incubate for 30 min at 4 °C in the dark.
5. Add 900 μL of PBS to each tube, mix by inverting, and centrifuge at 300 × g for 5 min at 4 °C. Discard the supernatant to remove excess antibodies.
6. Resuspend the cell pellet in each tube with 300 μL of PBS and analyze using a flow cytometer.
   NOTE: Cells do not need to be fixed with 2.5% neutral formaldehyde if the flow cytometer assay can be completed within 1–2 h. If analysis cannot be performed within 1–2 h, fix cells with 2.5% neutral buffered formalin (prepared by mixing 10% neutral buffered formalin with PBS in a 1:3 ratio) and store at 4 °C in the dark overnight for later flow cytometer assay.
7. Adjust the flow cytometer voltage using the label blank group to ensure cell autofluorescence is within approximately 1/4–1/3 of the coordinate axis. Sequentially load the cells from the CD29 isotype, CD29, CD44 isotype, CD44, Sca-1 isotype, Sca-1, CD31 isotype, CD31, CD45 isotype, and CD45 groups. Set the P1 gate (as defined in the FSC/SSC plot) to capture 10,000 cells, running at medium speed.
   NOTE: Ensure that the flow cytometer is equipped with the appropriate channels, such as those commonly used in the experiments: FITC, PE, PerCp-Cy5.5, PE-Cy7, and APC, or other channels that correspond to antibody-coupled fluoresceins.

8. Osteogenic differentiation of BMSCs

1. Resuspend P3 BMSC pellets in complete αMEM medium, adjusting the concentration to 1 × 10⁵ cells/mL.
2. Seed P3 BMSCs into a 24-well cell culture plate by adding 1 mL of complete αMEM medium containing 1 × 10⁵ cells to each well.
3. Culture the P3 BMSCs at 37 °C in a 5% CO₂ incubator for 3–5 days until they reach over 90% confluency. Replace the complete αMEM medium with fresh medium every 2 days.
4. Divide the different wells equally into two groups: the negative control group (without osteogenic differentiation induction) and the osteogenic differentiation group
5. Prepare the osteogenic differentiation induction medium by supplementing the complete αMEM medium with 10 mmol/L β-glycerophosphate, 50 mg/L L-ascorbic acid, and 10^-8 mol/L dexamethasone.
6. Discard the cell culture medium and wash the P3 BMSCs with 1 mL of PBS.
7. Add the osteogenic differentiation induction medium to the wells of the osteogenic differentiation group for the osteogenic differentiation induction (1 mL/well).
8. Add the complete αMEM medium to the wells of the control group (1 mL/well).
9. Culture the P3 BMSCs at 37 °C in a 5% CO₂ incubator for 7 or 21 days, replacing the osteogenic differentiation induction medium or the complete αMEM medium with the same fresh medium every 3 days.
   NOTE: Cells with a 7-day induction are used for the alkaline phosphatase (ALP) staining. Cells with a 21-day induction are used for the alizarin red S assay.

9. Identification of differentiated osteogenic cells with ALP staining

1. Wash the cells in each well of the 24-well plate with 1 mL of PBS once.
2. Fix the cells by adding 0.5 mL of 10% buffered formalin solution and incubating for 20 min.
3. Wash the cells in each well again with 1 mL of PBS once.
4. Add 0.5 mL of alkaline phosphatase staining reagent 1-Step NBT/BCIP to each well, and incubate at 37 °C for 30 min with aluminum foil wrapping to protect from light.
5. Discard the solution, wash the cells in each well with 1 mL of distilled water once, and allow the plate to dry.
6. Observe and acquire images of stained colonies using a camera to identify ALP-positive cells with black-purple precipitate.
   NOTE: The alkaline phosphatase (ALP) staining reagent 1-Step NBT/BCIP produces a strong, insoluble black-purple precipitate upon reaction with ALP, which can be easily visualized.

10. Identification of differentiated osteogenic cells by alizarin red S assay

1. Wash the cells in each well of the 24-well plate with 1 mL of PBS once.
2. Fix the cells by adding 0.5 mL of 10% buffered formalin solution and incubating for 20 min.
3. Wash the cells in each well of the 24-well plate with 1 mL of PBS once.
4. Add 0.5 mL of 0.1% alizarin red S in 0.1 mol/L Tris-HCl (pH 8.3) to each well, and incubate at 37 °C for 30–60 min.
5. Discard the solution, wash the cells in each well with 1 mL of distilled water once, and allow the plate to dry.
6. Observe and acquire images of stained colonies using a camera to identify alizarin red S-positive cells.
   NOTE: The alizarin red S assay for MSC differentiation into mature mineralized bone cells results in the formation of an alizarin red S-calcium complex, which produces a bright red stain that can be visually observed.

11. Preparation of adipogenic induction differentiation media A for BMSCs

NOTE: For adipocyte differentiation of BMSCs, a Mouse Bone Marrow Mesenchymal Stem Cells Adipogenic Differentiation Kit (see Table of Materials) is often used according to the guidelines.

1. Place the Fetal Bovine Serum (a component of the Kit, referred to as serum) in the 4 °C refrigerator for at least 6 h before complete thawing.
2. Place Supplement A-I (component of the Kit) in the 4° C refrigerator at least 30 min before preparation; place Supplement A-II (component of the Kit) at room temperature until fully thawed.
3. Invert or gently tap the reagent tube to mix the contents.
4. Centrifuge the Supplement A-II reagent tube at 300 × g for 30 s at room temperature to ensure the reagent collects at the bottom for easy retrieval.
5. Clean the outer packaging of all components thoroughly with 75% medical alcohol and open them in a biosafety cabinet.
6. Add serum, Supplement A-I, and Supplement A-II into the Medium.
   NOTE: To ensure proper dissolution, preheat the Basal Medium (a component of the Kit) to 37 °C, or Supplement A-II may precipitate due to cold exposure.
7. Use a small amount of Basal Medium to rinse each bottle and tube, making sure to transfer all components into the Basal Medium.
8. Secure the cap on the Basal Medium bottle and shake gently to mix thoroughly.
9. Seal the bottle with paraffin film, wrap it in aluminum foil, and label it with the name, preparation date, and other details.

12. Preparation of adipogenic induction differentiation media B for BMSCs

1. Place the Fetal Bovine Serum in the 4 °C refrigerator for at least 6 h before complete thawing.
2. Place Supplement B (a component of the Kit) in a 4 °C refrigerator until it is completely thawed for at least 30 min before preparation.
3. Invert or gently tap the reagent tube to mix the contents.
4. Centrifuge the Supplement B tube to concentrate the reagent at the bottom for easier collection.
5. Clean the outer packaging of all components thoroughly with 75% medical alcohol and open them in a biosafety cabinet.
6. Add serum and Supplement B into the Basal Medium.
7. Use a small portion of the Basal Medium to wash each bottle and tube, ensuring all components are fully transferred to the medium.
8. Secure the cap on the Basal Medium bottle and shake gently to mix thoroughly.
9. Seal the bottle with parafilm, wrap it in aluminum foil, and label it with the name, preparation date, and other details.

13. Adipocyte differentiation of BMSCs

1. Add 1 mL of 0.1% gelatin to a six-well plate, shaking it to evenly coat the bottom of each well.
2. Place the six-well plate coated with 0.1% gelatin in a sterile hood or CO₂ incubator for at least 30 min.
3. Aspirate the gelatin, and the plate is ready for cell seeding, or wait for the wells to dry before seeding after 30 min.
4. Seed the P3 BMSCs at a density of 4 × 10⁴ cells/cm² in each well of the six-well plate, adding 2 mL of complete medium to each well.
5. Incubate the cells at 37 °C in a CO₂ incubator with 5% CO₂ and saturated humidity.
6. Aspirate the medium carefully and add 2 mL of adipogenic differentiation induction medium A to each well when the cells reach 100% confluence.
7. Aspirate medium A from the wells after 3 days of induction, and add 2 mL of adipogenic differentiation medium B.
8. Aspirate medium B after 1 day, and switch back to medium A for further induction.
9. Alternate between medium A and B, and observe the cells daily. If cell contraction or death occurs during induction with medium A, switch to medium B promptly.
10. Repeat the induction and maintenance process until a sufficient number of lipid droplets of appropriate size appear, and then prepare for staining.

14. Oil Red O (ORO) staining

NOTE: Perform all procedures as gently as possible to avoid detachment of the lipid droplets.

1. Aspirate the adipogenic induction differentiation medium from the 6-well plate after the adipogenic induction differentiation is completed and gently wash with 1×PBS 2–3 times.
2. Add 2 mL of 10% neutral buffered formalin per well, and fix for 20 min at room temperature.
3. Prepare Oil Red O working solution by mixing Oil Red O stock with distilled water in a 3:2 ratio. After mixing, centrifuge at 250 × g for 4 min and use the supernatant.
4. Aspirate the fixative and gently wash with PBS 2–3 times, ensuring thorough removal of the fixative. 
5. Add 0.8 mL of red O stain solution to each well, leave at room temperature for 30 min.
6. Aspirate the stain solution, wash twice with 1 mL 75% ethanol.
7. Add 1 mL of hematoxylin counterstain to each well for 2 min, then aspirate.
8. Add 2 mL of PBS to each well and observe the adipogenic staining results under a microscope.
9. Seal the stained six-well plate with parafilm and store at 4 °C.
   NOTE: Do not store for more than 1 week. The lipid droplets may fuse, losing the staining state.

Results

Mouse-derived BMSCs were isolated and cultured for approximately 2 weeks to generate the third passage (P3) BMSCs (Figure 3). As shown in Figure 3A, cells freshly extracted from the bone marrow remained mononuclear with visible fat droplets. Spindle-shaped cells appeared in the culture dish, reaching a 60%-80% confluency on the 5^th culture day (Figure 3B,C). From the P1-P3...

Discussion

Stem cell therapy has garnered considerable interest due to its potential to revolutionize regenerative medicine. Preclinical studies have provided substantial evidence supporting the clinical application of stem cells, particularly BMSCs¹³. However, challenges such as low cell yield, contamination, and slow growth have impeded their clinical application. Therefore, there is an urgent need for research focusing on the standardization of BMSC preparation processes to enhance cell yield and function...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1mL syringe(22G syringe needle)	KINDLY GROUP(KDL)	60017031
1-Step NBT/BCIP	Pierce Biotechnology, Inc.	34042	Alkaline phosphatase staining
10% neutral buffered formalin	Wexis	5200250	cell fixation
6-well plate	Labselect	11110	Cell Culture
24-well plate	Labselect	11310	Cell Culture
40 μm Cell Strainer	Cornig	352340	Filtration to remove agglomerates
50 mL syringe	KINDLY GROUP(KDL)	60017040
75% ethanol	Shanghai NianYue Biotechnology Co., Ltd	31009001	Disinfect the skin of mice
Alizarin Red S	Sigma-Aldrich	A5533	Calcium Deposits and Calcified Nodules Detection
APC CD44 Monoclonal Antibody (clone:IM7)	eBioscience	17-0441-82	Flow cytometry
APC Rat IgG2b kappa Isotype Control (Clone:eB149/10H5)	eBioscience	17-4031-82	Flow cytometry
Autoclave	Japan Hiryama Company	HVE-50	Sterilization surgical instruments
CO2 incubator	Thermo Fisher Technology (China)Co., Ltd.	300583057	Cell Culture
Cytoflex flowmetry	Beckman Coulter	A00-1-1102	Flow cytometry
dexamethasone	Sigma-Aldrich	D4902	BMSCs osteogenic differentiation
Dish (100mm)	Corning	430167	Cell Culture
Dish (60mm)	Corning	430166	Sample placment during transplantation
Disinfectant cotton balls	Shanghai Honglong Industrial Co., Ltd.	20230627	Disinfect the skin of mice
Disposable sterile gloves	Shanghai Honglong Industrial Co.,Ltd	YT21131	Sterile operation
Double lion Irradiated Rodent Diet	Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd.	GB 14924.3	Animal feed
Fetal Bovine Serum	Shanghai Biosun Sci&Tech Co.,Ltd.	BS-0002-500	Cell Culture
FITC Hamster Anti-Rat CD29 (clone:Ha2/5)	BD Biosciences	555003	Flow cytometry
FITC Hamster IgM, λ1 Isotype Control (clone:G235-1)	BD Biosciences	553960	Flow cytometry
gelatin (0.1%)	OriCell	GLT-11301	enhance the adhesion ability of cells
IVCs mice cage	Suzhou Monkey King Animal Experimental Equipment Technology Co., Ltd.	HH-MMB-2	Animal barrier
L-ascorbic acid	Sigma-Aldrich	A8960	BMSCs osteogenic differentiation
Mark pen	Zebra Trading (Shenzhen) Co.,Ltd.	YYST5	Mark the cellular information on the Petri dish
MEM Alpha Modification (1X)	Shanghai NianYue Biotechnology Co., Ltd	SH30265	Cell Culture
Mouse Bone Marrow Mesenchymal Stem Cells Adipogenic Differentiation Kit	Cyagen	MUXMX-90031	BMSCs adipogenic differentiation
Nikon Eclipse Ti2 Inverted Microscope Synstem	Japan Nikon Corporatin	Ti2-U	Observe the cells and take photos
Ophthalmic scissors	Shanghai NianYue Biotechnology Co., Ltd	Y00030 JZ	Cut the skin
Ophthalmic tweezers	Shanghai NianYue Biotechnology Co., Ltd	BS-ZER-S-100 Biosharp	Obtain mouse bone tissue.
PASTEUR PIPETTE	Shanghai NianYue Biotechnology Co., Ltd	BS-XG-03M	Aspirate the culture medium
PE Rat Anti-Mouse CD31 (clone:Ha2/5)	BD Biosciences	553373	Flow cytometry
PE Rat IgG2a, κ Isotype Control (clone:R35-95)	BD Biosciences	553930	Flow cytometry
PE-Cyanine7 Ly-6A/E (Sca-1) Monoclonal Antibody (clone:D7)	eBioscience	25-5981-82	Flow cytometry
PE-Cyanine7 Rat IgG2a kappa Isotype Control (clone:eBR2a)	eBioscience	25-4321-82	Flow cytometry
Penicillin-Streptomycin	Shanghai NianYue Biotechnology Co., Ltd	2585636	Cell Culture
PerCP-Cy5.5 Rat Anti-Mouse CD45 (clone:Ha2/5)	BD Biosciences	561869	Flow cytometry
PerCP-Cy5.5 Rat IgG2b, κ Isotype Control (clone:A95-1)	BD Biosciences	550764	Flow cytometry
Scalpels	Surgical Instrument Factory oShanghai Medical Devices?Group?Co., Ltd.	J11010-10# JZ	Isolate the mouse's back limbs and torso, and remove extraneous tissues from the bone
Sterile hood	Thermo Fisher Technology (China)Co., Ltd.	ECO0.9	Surgical operation table
sterile surgical drapes	Henan Huayu Medical Equipment Co., Ltd.	20160900	Provide sterile surgery area
Tube (50 mL)	Shanghai Baisai, Biotechnology Co., Ltd.	BLD-BL2002500	Store the filtered cell solution
trypsin-EDTA (0.25%)	Gibco	25200056	digest adherent cells
β-glycerophosphate	Sigma-Aldrich	G5422	BMSCs osteogenic differentiation