Stemspan media

Manufactured by STEMCELL

Sourced in Canada

StemSpan media is a serum-free, specialized culture medium designed to support the expansion and maintenance of various types of stem cells, including hematopoietic, mesenchymal, and induced pluripotent stem cells. The core function of StemSpan media is to provide a defined, optimized environment for the in vitro culture of stem cells.

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Lab products found in correlation

Stem cell factor (scf), by R&D Systems (3 mentions) Heparin, by Merck Group (2 mentions) Low density lipoprotein, by Merck Group (1 mentions) Stemsep kit, by STEMCELL (1 mentions) Fibroblast growth factor 4 fgf4, by R&D Systems (1 mentions) Q vd oph qvd, by Merck Group (1 mentions) Qbsf 60 medium, by Quality Biological (1 mentions) Necrostatin 1 nec 1, by Selleck Chemicals (1 mentions) Buthionine sulfoximine bso, by Merck Group (1 mentions) Recombinant wnt5a, by R&D Systems (1 mentions) Human cd34 microbead kit, by Miltenyi Biotec (1 mentions) Automacs pro separator, by Miltenyi Biotec (1 mentions) Facscanto, by BD (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Sb216763, by Bio-Techne (1 mentions) 6 bromoindirubin 3 oxime bio, by Cayman Chemical (1 mentions) Sb415286, by Bio-Techne (1 mentions) Annexin 5 and 7 aad, by BD (1 mentions) Cd45 pe cy7, by BioLegend (1 mentions) Cd34 pacific blue, by BioLegend (1 mentions)

Close spelling variants of this product

StemSpan (104 citations) StemSpan SFEM media (21 citations) StemSpan SFEM II media (15 citations) StemSpan serum-free media (7 citations) StemSpan II media (5 citations) StemSpan SFEM serum free media (3 citations) StemSpan serum-free expansion media (3 citations) StemSpan SFEM II expansion media (2 citations) StemSpan Serum-Free Expansion Media (SFEM, (2 citations) StemSpan SFEM II human hematopoietic stem cell expansion media (2 citations)

21 protocols using stemspan media

Isolation and Expansion of MPSC from UCB

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Our method to produce MPSC from frozen samples of UCB is described in detail in other publications 12, 21, 25 and summarized here. We used either the Miltenyi‐MACS CD34+ selection kit, Bergisch, Germany or the Stem Cell Technologies Stem‐Sep kit, Vancouver, Canada to isolate CD34+ cells. CD34+ content was assessed using flow cytometry. The dead cell removal kit was used prior to CD34+ selection. Only frozen UCB units were used. Prior to the processing with the dead cell removal kit and selection, frozen units were filtered through a 70 micron mesh after thawing to remove clumps of dead cells that may have accumulated during the freeze/thaw process. Post column cells were seeded at 1 × 10⁵ cells/ml in FSFl medium (StemSpan media [Stem Cell Technologies] containing IMDM, 1% bovine serum albumin (BSA), 10 mg/ml insulin, 200 mg/ml human transferrin, 10⁻⁴ M 2‐mercaptoethanol, and 2 mM L‐glutamine. The media was supplemented with 25 ng/ml SCF [R&D Systems, Minneapolis, MN], 25 ng/ml Flt‐3 ligand [FL; R&D Systems, Minneapolis, MN] and 50 ng/ml Fibroblast Growth Factor‐4 [FGF‐4; R&D Systems, Minneapolis, MN], 50 ng/ml heparin and 10mg/ml low density lipoprotein [Sigma, Markham, Canada]). Fifty percent medium replacement occurred every 48 hours. For all animal experiments described here, the cells were used directly after 7–8 day culture in FSFl medium.

Whiteley J., Chow T., Adissu H., Keating A, & Rogers I.M. (2018). Topical Application of Culture‐Expanded CD34+ Umbilical Cord Blood Cells from Frozen Units Accelerates Healing of Diabetic Skin Wounds in Mice. Stem Cells Translational Medicine, 7(8), 591-601.

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Apoptosis assay in leukemia cell lines

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PDX cells were resuspended in QBSF-60 medium (Quality Biological, Gaithersburg, MD) with 20 ng/ml Flt-3 ligand, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine. Patient samples were cultured in StemSpan media (Stem Cell Technologies, Vancouver, Canada) with 20 ng/ml IL-3, 20 ng/ml IL-6, 100 ng/ml FLT-3L, 100 ng/ml SCF. The NALM6 cell line (RIKEN BioResource Center, Ibaraki, Japan) was used within 3 months of culture following validation by short tandem repeat analysis, and maintained in RPMI with 10% heat inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine. For some experiments cells were pre-treated with 10 ng/ml TNFα (Lonza Australia, Gordon, NSW), 10 μg/ml Enbrel (etanercept, soluble TNFR2 fused to Fc; Clifford Hallam Healthcare, VIC, Australia), 10 μM Q-VD-Oph (QVD; Sigma-Aldrich, St Louis, MO) or 10 μM Necrostatin-1 (Nec-1; Selleck Chemicals, Houston, TX) for 2 h before the addition of birinapant (TetraLogic Pharmaceuticals, Malvern, PA). Apoptosis was determined using Annexin V/7-AAD staining 24 h later.

Richmond J., Robbins A., Evans K., Beck D., Kurmasheva R.T., Billups C.A., Carol H., Heatley S., Sutton R., Marshall G.M., White D., Pimanda J., Houghton P.J., Smith M.A, & Lock R.B. (2016). Acute sensitivity of Ph-like acute lymphoblastic leukemia to the SMAC-mimetic birinapant. Cancer research, 76(15), 4579-4591.

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Isolation and culture of mouse hematopoietic stem cells

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For all experiments, lineage⁻, Sca-1⁺, c-kit⁺ (LSK) and LSK, CD150⁺, CD48⁻ cells were isolated as previously described [9 (link)]. Sorted LSK cells were cultured in serum-free StemSpan media (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 25 ng/mL stem cell factor (SCF) and fms-like tyrosine kinase-3 ligand (Flt3L). Sorted and LSK, CD150⁺, CD48⁻ cells were cultured in serumfree StemSpan media (supplemented with 25 ng/mL SCF and thrombopoietin (TPO). All cytokines were obtained from Peprotech (Rocky Hill, NJ, http://www.peprotech.com). Sorted cells were incubated overnight before reseeding at 5 × 10⁴ cells (LSK) or 1.5 × 10⁴ cells (LSK, CD150⁺, CD48⁻) per milliliter in fresh media. Recombinant Wnt5a (R&D Systems, Minneapolis, MN, http://www.abgent.com) and anti-Ryk antibody (clone RB1491, Abgent, San Diego, CA) were added at 250 ng/mL and 1 μg/mL, respectively. Media were changed at 48 hours. For specific experiments, cells were treated with 0.1 mM buthionine sulfoximine (BSO) (Sigma Aldrich).

Povinelli B.J, & Nemeth M.J. (2014). Wnt5a Regulates Hematopoietic Stem Cell Proliferation and Repopulation Through the Ryk Receptor. Stem cells (Dayton, Ohio), 32(1), 105-115.

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Isolation and Expansion of PBMCs

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Blood was delivered in heparin tubes. Fresh blood was diluted with phosphate buffered saline (PBS) and placed onto Ficoll-Paque reagent. The samples were centrifuged for 30 minutes at 850 ×g. Isolated PBMCs were transferred to a new tube and washed with PBS. Cell pellet was resuspended in StemSpan media (STEMCELL Technologies, Vancouver, Canada) containing CC110 cytokine (STEMCELL Technologies) for expansion. Cells were incubated for 5 days before reprogramming. Fresh media was added if needed.

Rim Y.A., Nam Y., Park N., Jung H., Jang Y., Lee J, & Ju J.H. (2018). Different Chondrogenic Potential among Human Induced Pluripotent Stem Cells from Diverse Origin Primary Cells. Stem Cells International, 2018, 9432616.

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Culturing Human Myeloid Leukemia Cell Lines

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The human myeloid leukemia cell lines U937 and KG1a were obtained from ATCC (Philadelphia, PA, United States) and were cultured in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS), L-glutamine, amphotericin B, and 1% penicillin-streptomycin maintained at 37°C and 5% CO₂. Cells were harvested during the log phase of growth.
Mononuclear cells obtained from bone marrows of AML patients (n = 5) were isolated using Ficoll-Hypaque. After red blood cell lysis, primary CD34⁺ BM cells were purified by magnetic bead separation using the human CD34 MicroBead kit and the AutoMACS Pro separator (Miltenyi Biotec). Purity of the CD34⁺ fraction was assessed by flow cytometry with anti-CD34-phycoerythrin (PE) antibody (BD Biosciences). CD34⁺ fractions showing purity greater than 90% were used. Cells were cultured in StemSpan media (Stem Cell Technologies) supplemented with 20% FBS, cytokines, including SCF, FLT-3L, TPO (all at 300 ng/mL), IL-3 (180 ng/mL), IL-6 (30 ng/mL), L-glutamine, amphotericin B, and 1% penicillin-streptomycin, maintained at 37°C with 5% CO₂.

Roversi F.M., Bueno M.L., Pericole F.V, & Saad S.T. (2021). Hematopoietic Cell Kinase (HCK) Is a Player of the Crosstalk Between Hematopoietic Cells and Bone Marrow Niche Through CXCL12/CXCR4 Axis. Frontiers in Cell and Developmental Biology, 9, 634044.

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Screening GSK-3 Inhibitors on BM-MNCs

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To screen GSK-3 inhibitors in vitro, 1 × 10⁵ BM-MNCs isolated by density centrifugation (Histopaque^® 1077, Sigma-Aldrich LLC, St. Louis, MO) were plated in StemSpan media (StemCell Technologies, Vancouver, BC, Canada) supplemented with 100 ng/mL stem cell factor (SCF) and 50 ng/mL thrombopoetin (TPO) as previously described (21 (link)). Cells were then irradiated with 3 Gy γ rays and 1 μM CHIR99021 (Stemgent, Cambridge, MA), 6-Bromoindirubin-3′-oxime (BIO) (Cayman Chemical, Ann Arbor, MI), SB415286 or SB216763 (Tocris Bioscience, Bristol, UK) was added to the culture wells 1 h after radiation exposure. Dimethylsulfoxide (DMSO) was used as vehicle to dissolve these compounds. The cells were incubated for 7 days using standard culture conditions and then counted and stained for phenotypic makers of HSPCs. HSPCs were analyzed based on surface marker expression of c-Kit and Sca1, and low to negative expression of lineage markers (anti-mouse CD3, CD4, CD8, B220, CD11b, Gr-1 and Ter-119 antibodies) using an antibody cocktail as previously described (22 (link)). All antibodies were purchased from BD Pharmigen or eBioscience. The cell analysis was performed on a FACSCanto (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc., Ashland, OR).

Lee C.L., Lento W.E., Castle K.D., Chao N.J, & Kirsch D.G. (2014). Inhibiting Glycogen Synthase Kinase-3 Mitigates the Hematopoietic Acute Radiation Syndrome in Mice. Radiation research, 181(5), 445-451.

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Apoptosis Analysis of BM-MNCs

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To analyze apoptosis, 1 × 10⁵ BM-MNCs isolated by density centrifugation (Histopaque 1077) were plated in StemSpan media (StemCell Technologies) supplemented with 100 ng/mL SCF and 50 ng/mL TPO and irradiated with 3 Gy. The cells were then treated with vehicle or SB216763 and cultured for 18 h before Annexin-V apoptosis assays were performed. The cultured cells were stained with KLS and then labeled with Annexin-V and 7-AAD (BD Biosciences) before analysis by flow cytometry.

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Investigating TC-S7005 Sensitivity in AML PDX

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All patient AML samples were collected with proper, informed consent and all experiments were performed according to an institutional review board-approved protocol, in accordance with the Declaration of Helsinki, and with an approved animal study IACUC protocol at Cincinnati Children’s Hospital Medical Center (CCHMC). Residual diagnostic specimens from AML patients at CCHMC were treated with an OKT3 antibody and engrafted into NSGS mice. Sensitivity of these PDX-derived AML cells was assessed in StemSpan media (STEMCELL Technologies; #09600) supplemented with 50 ng/mL human SCF, TPO, FLT3L, IL-3, and IL-6 with/without TC-S7005. The PDX-derived AML cells were plated in triplicate with the following doses of TC-S7005 (TOCRIS): 0 (DMSO), 0.5, 1, 5, and 10 μM.

Arimoto K.I., Miyauchi S., Troutman T.D., Zhang Y., Liu M., Stoner S.A., Davis A.G., Fan J.B., Huang Y.J., Yan M., Glass C.K, & Zhang D.E. (2023). Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis. Nature Communications, 14, 251.

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Mosaic Bone Marrow Transplant for M. avium Infection

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Whole bone marrow transplants were performed by IV injection of 2 × 10⁵ CD45.2 donor WBM cells from infected mice or naive controls with 2 × 10⁵ CD45.1 competitor into CD45.1 WT recipients following a split dose of 10.5 Gy of irradiation. For mosaic mice, lethally irradiated CD45.1 naïve recipient mice were transplanted with 1. 5 × 10⁵ unfractionated CD45.2 donor WBM (Dnmt3a^−/−, Dnmt3a^−/− Ifngr1^−/−, Batf2^−/−, or Cre-negative/WT) competed against 6 × 10⁵ unfractionated WBM cells from age-matched CD45.1 mice. Deletion of floxed alleles in Mx1-Cre Dnmt3a^f/f donor WBM was carried out 5–6 weeks before transplantation in primary recipients by six intraperitoneal injections (300 μg per mouse) of pIpC (Sigma) in PBS every other day. Eight weeks after transplantation, mosaic mice were infected with M. avium (or mock-infected with PBS) for eight additional weeks prior to bone marrow evaluation. To check the efficiency of deletion at the end of the experiment, CD45.2+ HSCs from mock-infected and infected mosaic mice were sorted into StemSpan media (Stem Cell Technologies), incubated for 2 weeks, and then the colonies were genotyped.

Hormaechea-Agulla D., Matatall K.A., Le D.T., Kain B., Long X., Kus P., Jaksik R., Challen G.A., Kimmel M, & King K.Y. (2021). Chronic Infection drives Dnmt3a-Loss of function Clonal Hematopoiesis via IFNg signaling. Cell stem cell, 28(8), 1428-1442.e6.

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Characterization of Hematopoietic Stem Cells

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Isolated CD34⁺ cells were propagated in round-bottom 96-well culture plates with no more than 5,000 cells per well under two different culturing conditions for 4 days: 1) undifferentiated, Stem Span Media supplemented with cytokine cocktail containing IL-3, IL-6, Flt, and SCF (Stem Cell Technologies) and 2) differentiated, EBM-2 media supplemented with EGM-2MV growth supplements (Lonza, Allendale, NJ) containing human epidermal growth factor, hydrocortisone, human fibroblast growth factor beta, VEGF, recombinant analog of insulin-like growth factor, ascorbic acid, heparin, and FBS.
At 4 days, the cells were stained with a cocktail of antibodies containing Pacific Blue CD34, PE/Cy7 CD45, FITC CD144, and PE CD133 (BioLegend). Cell-surface expression was analyzed using a BD LSR II Flow Cytometer, and the analysis was performed using FCS express (De Novo Software, Los Angeles, CA).

Bhatwadekar A.D., Yan Y., Stepps V., Hazra S., Korah M., Bartelmez S., Chaqour B, & Grant M.B. (2015). miR-92a Corrects CD34+ Cell Dysfunction in Diabetes by Modulating Core Circadian Genes Involved in Progenitor Differentiation. Diabetes, 64(12), 4226-4237.

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