For all studies, Lin- cells were enriched from total BM isolated from femurs and tibias of donor mice (6–12 weeks of age) using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Technologies) in conjunction with the RoboSep-S (STEMCELL Technologies) automated cell-isolation machine and confirmed by flow analysis. Cells were stained for lineage markers with a PE-Cy5-conjugated lineage marker cocktail including B220, Ter119, Tcrb, Cd8a, Cd3ε, Cd4, Ly6g/Ly6c, and Cd11b and with hematopoietic stem cell markers APC-conjugated anti-Cd117 (c-Kit) and PE-conjugated Ly6a/e (Sca-1) (BD Biosciences). Lin- cells were transduced with a lentiviral vector in a cell incubator at 2 × 106 cells/mL in either StemSpan SFEM II (STEMCELL Technologies) or StemMACS HSC Expansion Media (Miltenyi Biotec) growth media freshly supplemented with TPO (10 ng/mL), SCF (100 ng/mL), and FLT3 (50 ng/mL). Approximately 16–22 h later, cells were collected, washed at least three times with DPBS (without Ca2+ and Mg2+), and resuspended in an appropriate volume for dosing. A small number of cells (3,000–4,000 cells) were set aside for use in the colony-forming unit assay using M3434 media (STEMCELL Technologies) and were analyzed 7–10 days later.
Stemmacs hsc expansion media
Manufactured by Miltenyi Biotec
Sourced in United States
StemMACS HSC Expansion Media is a serum-free, animal component-free, and xenobiotic-free culture medium designed for the expansion of human hematopoietic stem and progenitor cells (HSPCs) in vitro. It provides the necessary nutrients and growth factors to support the proliferation and maintenance of the undifferentiated state of HSPCs.
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Lab products found in correlation
Robosep s, by STEMCELL (1 mentions)
Easysep mouse hematopoietic progenitor cell isolation kit, by STEMCELL (1 mentions)
Stemspan sfem 2, by STEMCELL (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Human cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
G csf, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by PAN Biotech (1 mentions)
Um729, by STEMCELL (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
K562 cells, by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by Harvard Bioscience (1 mentions)
Clinimacs system, by Miltenyi Biotec (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Calphos mammalian transfection kit, by Takara Bio (1 mentions)
4 protocols using stemmacs hsc expansion media
1
Lentiviral Transduction of Murine Hematopoietic Progenitors
2
Generation of t(4;11) Leukemia Cells
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CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from fresh human umbilical cord blood (huCB) obtained from the Department of gynecology from the University Hospital Tuebingen (IRB approvals 751/2015BO2 and 461/2022BO2) using Ficoll Paque (PAN-Biotech GmbH, Aidenbach, Germany) followed by the isolation of HSPCs with the human CD34+ Microbead Kit (Miltenyi, Bergisch Gladbach, Germany). t(4;11) was induced using an RNP complex of specific self-made sgRNAs and Cas9 protein (PNA Bio Inc., Thousand Oaks, CA, USA) as previously described [8 (link), 54 (link)]. A pure culture of t(4;11) cells was defined by more than 90% of translocated cells determined by Fluorescence-In-Situ-Hybridization (FISH) as previously described [54 (link)]. CD34+ and t(4;11) cells were maintained in StemMACS™ HSC Expansion Media (Miltenyi) supplemented with 10% FCS (Gibco by Thermo Fisher Scientific, Waltham, MA, USA), 1% Penicillin/Streptomycin (Lonza, Basel, Switzerland), human cytokines (IL-3, IL-6, SCF, FLT3L, SCF, G-CSF, 50 ng/ml each, PeproTech, Rocky Hill, NJ, USA), SR-1 and UM-729 (0.75 µM each, STEMCELL Technologies, Vancouver, Canada).
3
Expansion of Human Hematopoietic Stem Cells
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K-562 cells were acquired from Sigma-Aldrich and cultured at 37 °C with 5% CO2 in RPMI (Biochrom) supplemented with 10% FBS (Gibco), 1% L-glutamine (Biochrom), and 1% Penicillin/Streptomycin (Biochrom).
Immunomagnetic enrichment of HSPCs was performed using magnetic-activated cell sorting system (CliniMACS System, Miltenyi Biotec), according to the manufacturer’s instructions. CD34+ HSPCs were then cultured at 37 °C with 5% CO2 in StemMACS HSC Expansion Media (Miltenyi Biotec) supplemented with human cytokines (Miltenyi Biotec): SCF (100 ng/ml), TPO (20 ng/ml), and Flt3-L (100 ng/ml).
Immunomagnetic enrichment of HSPCs was performed using magnetic-activated cell sorting system (CliniMACS System, Miltenyi Biotec), according to the manufacturer’s instructions. CD34+ HSPCs were then cultured at 37 °C with 5% CO2 in StemMACS HSC Expansion Media (Miltenyi Biotec) supplemented with human cytokines (Miltenyi Biotec): SCF (100 ng/ml), TPO (20 ng/ml), and Flt3-L (100 ng/ml).
4
Lentiviral Vector Production for WT1-TCR
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HIV-derived self-inactivating third-generation lentiviral vectors were constructed using the CMV (Cytomegalovirus) enhancer and promoter driving expression of the viral transcript (pCCL plasmid backbone).9 12–14 (link) A WT1-specific codon-optimized TCR sequence15 (link) was cloned into the lentiviral transfer vector plasmid under control of the myeloproliferative sarcoma virus enhancer (MND) promoter.16 (link) Lentiviral vectors were produced by transient cotransfection of HEK293T cells with the lentiviral transfer vector plasmid (LV.WT1-TCR) and respective packaging plasmids (pRSV-Rev, pMDLg/pRRE, and pMD2-VSV-G) using CalPhos Mammalian Transfection Kit (Clontech), as previously described.9 (link) Viral supernatants were filtered through 0.45 µm low-protein binding filters, concentrated by ultracentrifugation at 20 000 rpm for 2 hours, resuspended in StemMACS HSC Expansion Media (Miltenyi Biotec) and stored at −80°C. Vesicular stomatitis virus (VSV)-G protein pseudotyped lentiviral particles were titrated on Jurkat cells. Concentrated viral vector supernatants were serially diluted on Jurkat cells. At 72 hours post-transduction, cells were harvested and analyzed by flow cytometry for expression of TCRVβ21.3, recognizing the recombinant WT1-TCR, to determine viral titer.
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