Bone marrow cells were obtained from the crushed bones of ∼4- to 6-mo-old mice and immunostained and sorted for Lin+CD45+ cells (BMLs) or magnetically enriched by cKit and then immunostained for HSCs, MPPs, and OPPs (Fig. S2 A ). Bone marrow mesenchymal stromal cells were harvested from mouse bone marrow (Ding et al., 2012 (link)), immunostained, negatively enriched (CD45−, TER119−) using EasySep mouse mesenchymal stem/progenitor cell enrichment kit following vendor’s instructions (catalog no. 19771; StemCell Technologies), and sorted based on PDGFRα expression. FACS sorting was performed with a BD FACS Aria II cell sorter (BD Biosciences) at 4°C.
Easysep mouse mesenchymal stem progenitor cell enrichment kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit is a magnetic cell separation system used to enrich mesenchymal stem/progenitor cells from mouse samples. The kit utilizes antibody-coated magnetic particles to isolate the target cells from the sample, allowing for a quick and easy separation process.
Automatically generated - may contain errors
Lab products found in correlation
Mesencult mouse msc proliferation medium, by STEMCELL (2 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Coulter act diff analyzer, by Beckman Coulter (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Percoll density gradient centrifugation method, by GE Healthcare (1 mentions)
Collagenase, by Fujifilm (1 mentions)
Facsaria 2, by BD (1 mentions)
6 protocols using easysep mouse mesenchymal stem progenitor cell enrichment kit
1
Isolation and Sorting of Murine Bone Marrow Cells
2
Isolation and Characterization of Murine Bone Marrow and Splenic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BM was flushed from femurs using phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). Spleens were harvested from mice and processed in PBS and 2% FBS using the Miltenyi gentleMACS and a C-type tube (Bergisch Gladbach, Germany). Blood was collected via cardiac puncture into 3.2% sodium citrate. Each fraction was counted using a Coulter AcT Diff Analyzer (Beckman Coulter).
To isolate BM stromal/endothelial cells, bones were harvested from NSG and C57BL/6 mice. BM was flushed and discarded and the bones were treated with 1 mg/mL collagenase type-1 (Worthington) as previously described.23 (link) Blood cells were depleted using the EasySep™ Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (Cat. n. 19771 StemCell Technologies) following the manufacturer’s protocol.
BM-MSC were isolated from NSG femurs using a modification of a previously described protocol24 (link) (see Online Supplementary Methods).
To isolate BM stromal/endothelial cells, bones were harvested from NSG and C57BL/6 mice. BM was flushed and discarded and the bones were treated with 1 mg/mL collagenase type-1 (Worthington) as previously described.23 (link) Blood cells were depleted using the EasySep™ Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (Cat. n. 19771 StemCell Technologies) following the manufacturer’s protocol.
BM-MSC were isolated from NSG femurs using a modification of a previously described protocol24 (link) (see Online Supplementary Methods).
3
Isolation and Characterization of Murine Adipose-Derived Mesenchymal Stem Cells
4
Isolation and Enrichment of Murine Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adipose tissue was collected from the abdominal and inguinal regions from wild type (WT) mice, incubated with 0.1% type I collagenase solution in a 195‐rpm shaker at 37°C for 90 minutes, centrifuged at 300g for 5 minutes, shaken vigorously for 15 seconds and centrifuged at 300g for an additional 5 minutes at room temperature. The dark cell pellets were collected, suspended in phosphate buffer saline (PBS) containing 10% bovine serum albumin (BSA) and centrifuged at 300g for 5 minutes. The cell pellets were then suspended in cold 1× Magcellect plus via a negative selection principle (CD45‐, TER119‐; EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit, Stem Cell Technologies, Vancouver, Canada, https://www.stemcell.com/ ). The cells were maintained in Mesencult mouse MSC proliferation medium (Stem Cell Technologies Inc., Vancouver, BC, Canada, https://www.stemcell.com/ ) and used at passage 2. These cells were 99.99% CD45 negative and positive for CD105 (>70%), CD29 (>99%) and Sca1 (>98%) 46 .
5
Isolation and Characterization of Human and Mouse Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human MSCs (hMSCs) were derived from bone marrow samples from 5 healthy premenopausal women (41–46 years old) and 5 women with postmenopausal osteoporosis (51–59 years old). Informed consent was obtained from each participant as a donor of bone marrow. Osteoporosis was diagnosed using the World Health Organization parameters. The hMSCs were purified from the bone marrow samples according to the Percoll density gradient centrifugation method (GE Healthcare Life Sciences). The primary mouse bone marrow cells were collected from the tibia and femur by crushing the bones, then the mouse MSCs (mMSCs) were isolated and purified using EasySep™ Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (StemCell). Both hMSCs and mMSCs were cultured in a modified essential medium (a-MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/mL streptomycin in an incubator with a humidified atmosphere and 5% CO2 at 37°C. The hMSCs and mMSCs, which were positive (>95%) for CD29, CD44, CD90, and CD105 but were negative (<5%) for hematopoietic markers CD34, CD14, and CD45, were used for further study.
HEK-293T cells were obtained from ATCC and were cultured in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin in an incubator with a humidified atmosphere and 5% CO2 at 37°C.
HEK-293T cells were obtained from ATCC and were cultured in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin in an incubator with a humidified atmosphere and 5% CO2 at 37°C.
6
Isolation of Murine Bone Marrow-Derived Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To isolate MSCs, BM cells were first excluded from the femurs and tibias of the mice by flushing. The bones were then crushed with a pestle and treated with collagenase (Wako, Osaka, Japan), after which MSCs were separated using the EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (StemCell Technologies, Vancouver, British Columbia, Canada) to separate mesenchymal stromal cell fraction (defined by Ter119 -CD45 -). BM-MSC fractions, defined by Ter119 -CD45 -CD31 -Sca-1 + , were sorted by BD FACSAria II flow cytometers (BD Biosciences, Franklin Lakes, NJ, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!