Primary human CD34+ HSPCs (STEMCELL Technologies) were grown in StemSpan™ SFEM with 1% StemSpan™ CC110, 0.5% L-glutamine, and 0.5% penicillin-streptomycin in a humidified atmosphere of 5% CO2/95% air at 37°C. For colony-forming unit (CFU) assays, CD34+ HSPCs were cultured for 18 days in StemMACS™ HSC-CFU complete with Epo (Miltenyi Biotec), and hematopoietic progenitor CFUs were quantitated and classified according to morphology.
Stemspan cc110
Manufactured by STEMCELL
Sourced in Canada
The StemSpan CC110 is a fully automated cell culture system designed for the expansion and differentiation of human and mouse stem and progenitor cells. It provides a controlled and closed environment for cell culture, with precise control over culture parameters such as temperature, gas composition, and media replenishment.
Automatically generated - may contain errors
Lab products found in correlation
Tesr e8 medium, by STEMCELL (2 mentions)
Stemspan sfem 2 media, by STEMCELL (2 mentions)
Stemspan sfem, by STEMCELL (1 mentions)
Vitronectin coated plates, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus media, by GE Healthcare (1 mentions)
Stemspan acf, by STEMCELL (1 mentions)
Biocoat, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Cyclosporine h, by Merck Group (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Poloxamer, by BASF (1 mentions)
4d nucleofector, by Lonza (1 mentions)
P3 primary cell kit, by Lonza (1 mentions)
5 protocols using stemspan cc110
1
Hematopoietic Progenitor Cell Culture and CFU Assay
2
iPSC Generation from Human PBMCs
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Peripheral blood samples were obtained from healthy human donors after obtaining informed consent, with approval of the Institutional Review Board of Seoul St. Mary’s Hospital (KIRB-00613_6-001). Peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation using Ficoll-Paque PLUS media (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Isolated PBMCs were cultured in StemSpan ACF (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with StemSpan CC110 for 4 days. Mononuclear cells were then transferred to a 24-well vitronectin-coated plate (BD BioCoat, BD Biosciences, Franklin Lakes, NJ, USA), and Sendai virus (SeV) (CytoTune-iPSC 2.0 Reprogramming kit, Thermo Fisher Scientific, Waltham, MA, USA) was added to give a multiplicity of infection of three (MOI:3). The medium was changed daily until iPSC colonies formed. After manual picking, iPSC lines were maintained on vitronectin-coated plates (Thermo Fisher Scientific) in a TeSR-E8 medium (Stem Cell Technologies). Emerging iPSC colonies were picked individually and expanded for characterization on day 12 after transduction. Cells were cultured in a 37°C incubator with 5% CO2 from days 3 to 21 after transduction.
3
Generating iPSCs from FDN Patient PBMCs
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Peripheral blood mononuclear cells (PBMCs) were obtained from a patient with FDN. We generated hiPSCs (CMC-Fb-001 and CMC-Fb-003) as described in previous studies [19 –22 (link)]. Briefly, PBMCs were isolated by centrifugation using Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA) and cultured in StemSpa ACF (Stem Cell Technologies, Vancouver, Canada) supplemented with StemSpan™ CC110 for 4 days. Mononuclear cells were then transferred to a 24-well plate manually coated with recombinant human vitronectin (BD BioCoat). Sendai virus (SeV) (CytoTune-hiPSC 2.0 Reprogramming Kit; Life Technologies, Invitrogen, Carlsbad, CA, USA) was then added at a multiplicity of infection of three. The medium was changed daily until hiPSC colonies formed. After manual picking, hiPSC lines were maintained on vitronectin (Life Technologies, Invitrogen)-coated plates in TeSR-E8 medium (Stem Cell Technologies). On day 12 after transduction, emerging hiPSC colonies were picked individually and expanded for characterization. From day 3 to day 21 after transduction, cells were cultured in a 37 °C incubator with 5% CO2. We regularly checked the existence of mycoplasma in cells, which showed no positive results. (Additional file 1 : Fig. S1).
4
Transduction of CD34+ Cells with Cas9-EDVs
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Cryopreserved G-CSF-mobilized human CD34 + cells were acquired from AllCells. Cells were thawed, resuspended in IMDM (Gibco), spun and then cultured in StemSpan SFEM II media (StemCell Technologies) with 100 U ml -1 penicillin-streptomycin (Gibco) and the cytokine https://doi.org/10.1038/s41587-023-02085-z cocktail StemSpan CC110 (StemCell Technologies). Cells were treated with 8 μM cyclosporine H (Sigma-Aldrich) for 24 h before treatment. Additionally, cells were treated with 1 μg ml -1 poloxamer (BASF) at the time of treatment. Cas9-EDVs were concentrated 50-fold using Lenti-X Concentrator (Takara Bio), resuspended in SFEM II media and mixed with 40,000 CD34 + cells in a final well volume of 100 μl. Transduction was performed in a U-bottom 96-well plate for 24 h on an orbital shaker before cells were spun, expanded 1:1 and cultured stationary until analysis.
5
Genetic Modification of Human HSCs
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Mobilized peripheral blood CD34+ cells were acquired from AllCells and cultured in StemCell StemSpan™ SFEM II media (StemCell No. 09605) supplemented with StemSpan CC110 (StemCell No. 02697) cytokine cocktail for 48 h before nucleofection. For gene-editing experiments, RNPs were formed as described previously and electroporated into 200,000 HSCs using the P3 Primary Cell kit (Lonza No. V4XP-3032) in the Lonza 4D nucleofector using the CA-137 program. Following electroporation, HSCs were resuspended in 1 mL of culture medium and cultured in 24-well plate format. For experiments using AAV-6 transduction, the relevant donor AAV-6 at the indicated MOI was diluted in culture media before electroporation, and added to cells immediately after the electroporation event. Seventy-two hours postelectroporation, cells were harvested for flow cytometry and genomic DNA extraction as described previously.
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