Stemspan sfem 2 medium

Manufactured by STEMCELL

Sourced in Canada

StemSpan SFEM II is a serum-free, feeder-free culture medium designed to support the expansion and maintenance of human hematopoietic stem and progenitor cells.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (6 mentions) Matrigel, by Corning (5 mentions) Il 6 cytokines, by Thermo Fisher Scientific (4 mentions) Cytotune ips 2.0 sendai reprogramming vectors, by Thermo Fisher Scientific (4 mentions) Y 27632, by STEMCELL (4 mentions) Histopaque 1077, by Merck Group (4 mentions) Mtesr1 medium, by STEMCELL (4 mentions) Dispase, by STEMCELL (4 mentions) Cryostor cs10 freezing medium, by STEMCELL (4 mentions) Stemspan erythroid expansion supplement, by STEMCELL (4 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (4 mentions) L glutamine, by Thermo Fisher Scientific (4 mentions) Hflt3l, by Thermo Fisher Scientific (3 mentions) Human cd34 microbead kit, by Miltenyi Biotec (2 mentions) Um729, by STEMCELL (2 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Um171, by Selleck Chemicals (2 mentions) Rosettesep human progenitor cell basic pre enrichment antibody cocktail, by STEMCELL (2 mentions) Stemspan myeloid expansion supplement, by STEMCELL (2 mentions)

Close spelling variants of this product

StemSpan™ Serum-Free Expansion Medium II (SFEM II) StemSpanTM SFEM II serum-free medium StemSpan SFEM medium StemSpan SFEM II StemSpan SFEM SFEM II medium StemSpan II medium SFEM medium StemSpan medium StemSpan II

49 protocols using stemspan sfem 2 medium

Expansion of Primary NK Cells from Peripheral Blood and Cord Blood Hematopoietic Stem Cells

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Peripheral blood mononuclear cells (PBMCs), procured from the peripheral blood of healthy donors, were segregated using density gradient centrifugation facilitated by Ficoll-Hypaque (1.077 g/mL). To facilitate PB-NK cell expansion, thawed PBMCs were combined with 2 × 10⁵ K562-mbIL21-feeder cells at a proportion of 1 × 10⁵ cells per ml in StemSpan™ SFEM II medium (Stemcell Technologies). The medium was enriched with 1% L-glutamine (Invitrogen), 100 ng/ml hIL-2 (Peprotech), 20 ng/ml hIL-7 (Peprotech), 20 ng/ml hIL-15 (Peprotech), and 50 μg/ml ascorbic acid (Sigma) [20 (link)]. Both the cytokines and ascorbic acid were freshly added prior to use.
Cord blood CD34⁺ HSPCs were isolated using the CD34 MicroBead Kit (Miltenyi Biotec) [21 (link)]. The enriched HSPC population comprised over 90% CD34⁺ cells. These HSPCs were introduced at 5 × 10⁵ cells per ml into serum-free StemSpan™ SFEM II medium (Stemcell Technologies). The medium was enriched with 1% L-glutamine (Invitrogen), 100 ng/ml hSCF (Peprotech), 100 ng/ml hFlt3-L (Peprotech), 100 ng/ml hTPO (Peprotech), 50 ng/ml hIL-6 (Peprotech), 750 nM SR1 (Sigma), and 50 nM UM171 (Sigma). To maintain optimal cell density, fresh medium was administered every two days before initiating NK cell differentiation, thus ensuring a cell density range of 5 × 10⁵ to 1 × 10⁶ cells per ml.

Wen W., Chen X., Shen X.Y., Li H.Y., Zhang F., Fang F.Q, & Zhang X.B. (2023). Enhancing cord blood stem cell-derived NK cell growth and differentiation through hyperosmosis. Stem Cell Research & Therapy, 14, 295.

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Erythroid Differentiation of Cord and Peripheral Blood CD34+ Cells

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Primary CD34⁺ mononuclear cells derived from cord blood and peripheral blood (StemCell Technologies) were expanded for up to 7 days in StemSpan SFEM II medium (StemCell Technologies) supplemented with StemSpan CC100 (1×, StemCell Technologies) and penicillin/streptomycin (2%, Life Technologies). To induce erythroid differentiation, cells were cultured for up to 10 days in StemSpan SFEM II medium (StemCell Technologies) supplemented with SCF (10 ng/ml, Invitrogen), EPO (5 U/ml, Invitrogen), IL-6 (10 ng/ml, Sigma) and penicillin/streptomycin (2%, Life Technologies).

Morrison T.A., Wilcox I., Luo H.Y., Farrell J.J., Kurita R., Nakamura Y., Murphy G.J., Cui S., Steinberg M.H, & Chui D.H. (2017). A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression. Blood cells, molecules & diseases, 69, 1-9.

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Erythroid Differentiation of Cryopreserved CD34+ Cells

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Cryopreserved human bone marrow CD34⁺ cells were purchased from STEMCELL Technologies (Catalog No. 70002.3; Vancouver, Canada). Frozen bone marrow CD34⁺ cells were thawed, washed once with StemSpan™ SFEM II medium (Catalog No. 09605; STEMCELL Technologies), suspended in StemSpan™ SFEM II medium containing StemSpan™ Erythroid Expansion Supplement (Catalog No. 02692; STEMCELL Technologies), and cultured for 7 days. CD34⁺ cells were then harvested, washed once with Dulbecco's (D)‐PBS, and suspended at 0.5 x 10⁶ cells/mL in human whole blood to be used in the EPO stimulation assay.
Human whole blood was collected from 13 healthy donors (seven males and six females) via venipuncture into Vacutainer collection tubes containing sodium heparin, in accordance with Pfizer protocols (Protocol No. GOHW RDP‐01) approved by the Shulman Institutional Review Board. Blood was warmed to 37°C prior to use.

Dowty M.E., Lin T.H., Jesson M.I., Hegen M., Martin D.A., Katkade V., Menon S, & Telliez J. (2019). Janus kinase inhibitors for the treatment of rheumatoid arthritis demonstrate similar profiles of in vitro cytokine receptor inhibition. Pharmacology Research & Perspectives, 7(6), e00537.

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Mouse Hematopoietic Stem Cell Culture

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Freshly isolated sorted LSKs from mice were plated in StemSpan medium SFEM II (STEMCELL Technologies) with additional ingredients (50 ng/ml SCF, 50 ng/ml TPO, 50 ng/ml FL3 ligand, and 50 ng/ml IL-3) at 37°C in a humidified atmosphere of 5% CO₂. LSKs were seeded at 2000–3000 cells per well in the presence of vehicle (DMSO) or inhibitors (all purchased from Selleck) with several concentrations in SFEM (STEMCELL Technologies) media. Relative and absolute numbers of LSKs were analyzed after 7 days of culture by flow cytometry. Human embryonic kidney 293T cells were cultured in DMEM medium with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂.

He Q., Hong M., He J., Chen W., Zhao M, & Zhao W. (2019). Isoform-specific involvement of Brpf1 in expansion of adult hematopoietic stem and progenitor cells. Journal of Molecular Cell Biology, 12(5), 359-371.

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Lentiviral Transduction of Human HPCs

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Mononuclear cells from cord blood were isolated via a Ficoll-Paque PLUS (GE Healthcare) gradient. Mononuclear cells were washed and resuspended in FACS buffer (PBS without Ca²⁺ and Mg²⁺, supplemented with 2% heat-inactivated FBS and 5 mM EDTA). CD34⁺ cells were purified using a Human CD34 MicroBead kit from Miltenyi Biotec (130-100-453).
CD34⁺ human HPCs were resuspended at a concentration of 1 million cells per ml in StemSpan medium SFEM II (Stemcell Technologies, 09605) supplemented with 100 ng ml⁻¹ SCF, 100 ng ml⁻¹ FLT3L and 50 ng ml⁻¹ TPO (so-called stem cell medium). Two hundred thousand cells were plated in a 48-well plate, and 5 μl lentiviral construct (either BRAF^V600E or NGFR) was added to the medium at an MOI of 50. Cells were incubated at 37 °C with 5% CO₂. Supplemented StemSpan medium was added 24 h after the initiation of transduction, and cells were kept in culture for up to 14 d. GFP-transduced cells were analyzed for myeloid and lymphoid surface marker expression (see antibody list) starting 2 d after transduction.

Bigenwald C., Le Berichel J., Wilk C.M., Chakraborty R., Chen S.T., Tabachnikova A., Mancusi R., Abhyankar H., Casanova-Acebes M., Laface I., Akturk G., Jobson J., Karoulia Z., Martin J.C., Grout J., Rafiei A., Lin H., Manz M.G., Baccarini A., Poulikakos P.I., Brown B.D., Gnjatic S., Lujambio A., McClain K.L., Picarsic J., Allen C.E, & Merad M. (2021). BRAFV600E-induced senescence drives Langerhans cell histiocytosis pathophysiology. Nature medicine, 27(5), 851-861.

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Generation of induced pluripotent stem cells

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PBMCs were isolated from the peripheral blood sample using Histopaque®−1077 (Sigma-Aldrich) and cultured in StemSpan SFEMIImedium (Stem Cell Technologies) supplemented with 100 ng/mL SCF, 100 ng/mL FLT-3 L, 20 ng/mL IL-3 and 20 ng/mL IL-6 cytokines (Peprotech). Five days later, the cells were counted and transduced using CytoTune®-iPS 2.0 Sendai reprogramming vectors (Thermo Fisher) following the manufacturer’s instruction. The transduced cells were then plated onto irradiated mouse embryonic fibroblasts (MEFs) and cultured in mTeSR™1 medium (Stem Cell Technologies) which was changed every other day. Around day19 post-transduction, ESC-like colonies appeared and were manually picked on day25 post-transduction. The iPSCs were cultured on Matrigel (Corning)-coated plates in mTeSR™1 medium at 37 °C with 5% CO₂ and routinely passaged at 1:3 ratio using dispase (Stem Cell Technologies) every 4–6 days. The iPSCs were frozen in CryoStor® CS10 freezing medium and thawed with 10 μM Y-27632 (Stem Cell Technologies). hESC (H7 [Wi Cell Research Institute, Madison, WI, USA]) was cultured in parallel with FDEENTi002-A.

Bai X., Yang X.J, & Chen L. (2019). Establishment of an induced pluripotent stem cell line (FDEENTi002-A) from a patient with Best’s disease carrying c.888C > A mutation in BEST1 gene. Stem cell research, 38, 101459.

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Expansion and Maintenance of HUDEP2 and Tet-On-Cas9 iPSCs

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HUDEP2 (HUDEP-2) cells (a kind gift from Yukio Nakamura) (Kurita et al., 2013 (link)) were cultured following previously reported protocols (Kurita et al., 2013 (link); Bagchi et al., 2021 (link)). Briefly, the cells were expanded in StemSpan SFEM-II medium (Stem Cell Technologies) supplemented with 1 µM dexamethasone, 1 μg/mL doxycycline (Dox), 50 ng/mL recombinant human stem cell factor (rh SCF), 3 units/mL recombinant human erythropoietin (rh EPO), 10 ng/mL recombinant human interleukin 3 (rh IL-3) and 1% penicillin/streptomycin. The AAVS1-Tet-On-Cas9 iPSCs generated in our laboratory (Thamodaran et al., 2022 (link)) were cultured in Matrigel (Corning) - coated plates containing mTeSR medium (STEMCELL Technologies). Upon reaching 70%–80% confluency, colonies were dissociated with Versene (Thermo Fisher Scientific) following the manufacturer’s protocol and passaged at a 1:4 ratio.

Ijee S., Chambayil K., Chaudhury A.D., Bagchi A., Modak K., Das S., Benjamin E.S., Rani S., Paul D.Z., Nath A., Roy D., Palani D., Priyanka S., Ravichandran R., Kumary B.K., Sivamani Y., S. V., Babu D., Nakamura Y., Thamodaran V., Balasubramanian P, & Velayudhan S.R. (2024). Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs. Frontiers in Molecular Biosciences, 10, 1295507.

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Expansion of Human CD34+ Hematopoietic Stem Cells

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Human CD34+ HSPCs were obtained from healthy male and female volunteers (age, 31–60 years) after informed consent in accordance with the Declaration of Helsinki and under an institutional review board-approved clinical protocol (https://clinicaltrials.gov/ct2/show/NCT00001529; ClinicalTrials.gov: NCT00001529). Peripheral blood mobilization of HSPCs was induced via subcutaneous injection of 10 μg/kg granulocyte-colony stimulating factor (G-CSF, Filgrastim, Amgen) for 5 days, followed by leukapheresis using a Cobe Spectra Apheresis System (Terumo BCT). The mononuclear cell concentrates were enriched in CD34+ HSPCs using a CliniMACS Plus instrument (Miltenyi Biotec) and cryopreserved prior to use. All CD34+ HSPCs were cultured in StemSpan SFEM II medium (STEMCELL Technologies) supplemented with stem cell factor (SCF; 100 ng/mL), thrombopoietin (TPO; 100 ng/mL), Fms-like tyrosine kinase 3 ligand (Flt3-ligand; 100 ng/mL), UM729 (0.8 μM, STEMCELL Technologies), and 1% penicillin-streptomycin (GIBCO). Cells were cultured at 37°C, 5% CO₂, and 5% O₂.

Bloomer H., Smith R.H., Hakami W, & Larochelle A. (2020). Genome editing in human hematopoietic stem and progenitor cells via CRISPR-Cas9-mediated homology-independent targeted integration. Molecular Therapy, 29(4), 1611-1624.

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Hematopoietic Stem Cell Genome Editing

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Human bone marrow CD34⁺ hematopoietic stem/progenitor cells were obtained from AllCells and Fred Hutchinson Cancer Research Center. CD34⁺ cells were maintained in StemSpan SFEM II medium supplemented with CC110 (StemCell Technologies). K562 cells were cultured in IMDM supplemented with 10% FBS and 1% penicillin/streptomycin. Frozen stocks were prepared with 10% DMSO and 30% FBS in K562 medium and stored in liquid nitrogen prior to use. Genome editing experiments in hESCs were performed as previously described (Hockemeyer et al,2009; Soldner et al,2009; Hockemeyer et al,2011) in the WIBR#3 line (Lengner et al,2010) and NIH stem cell registry # 0079. Cell culture was carried out as previously described in Boyle et al (2020).

Kim W., Hennick K., Johnson J., Finnerty B., Choo S., Short S.B., Drubin C., Forster R., McMaster M.L, & Hockemeyer D. (2021). Cancer‐associated POT1 mutations lead to telomere elongation without induction of a DNA damage response. The EMBO Journal, 40(12), e107346.

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Isolation and Culture of CD34+ HSPCs

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Primary human CD34^pos-HSPCs isolated from umbilical cord blood (CD34^pos-HSPCs) and the mononuclear cells (MNC) from whole cord blood were purchased from ALL Cells (USA). For CD34^pos-HSPCs isolation from whole cord blood, the MNCs were washed and filtered through a 100 μm cell strainer (BD Falcon) followed by lineage depleton by negative selection using a lineage cell depletion cocktail (Milteny Biotec). The lineage depleted fraction was stained with 13 markers for lineage-specfic monoclonal antibodies FITC (CD2, CD3, CD4, CD11b, CD14, CD15, CD24, CD10, CD41, CD20, CD66c, CD127, CD33) (Biolegend and BD) and Pacific blue conjugated anti-CD34 (Biolegend), for 30 min at 4 °C. Before sorting, 7-AAD was added to the tubes. Afterwards, 13Lin−CD34^pos cells were collected by the fluorescence-activated cell sorter (FACSAria, BD Biosciences, San Jose, CA, USA).
For CD34^pos-HSPCs, the cells were thawed using stem span SFEM II medium (Stem Cell Technologies, Canada) and washed 1× with the same medium. The cells were resuspended in 1 mL of medium contained 1× Stem Span Cytokine Cocktail CC100 (Stem Cell Technologies, Canada) in 24-well plate, then the cells were analyzed within 24 h.

Al Alwan B., AbuZineh K., Nozue S., Rakhmatulina A., Aldehaiman M., Al-Amoodi A.S., Serag M.F., Aleisa F.A., Merzaban J.S, & Habuchi S. (2021). Single-molecule imaging and microfluidic platform reveal molecular mechanisms of leukemic cell rolling. Communications Biology, 4, 868.

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