Lin− mouse BM cells were isolated by flushing femurs, tibias, and iliac crests of 6- to 8-week-old CD45.2 C57BL/6 or CD45.2 Berkeley SCD mice (BERK-SCD, JAX stock #003342) followed by lineage depletion using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin− cells were pre-stimulated at 1 × 106 cells/mL in Stem Cell Growth Medium (CellGenix) supplemented with mouse SCF (100 ng/mL), hTPO (100 ng/mL), mouse IL-3 (mIL-3) (20 ng/mL), and hFlt3-L (100 ng/mL), all from Peprotech. Following a 36- 40-h pre-stimulation, cells were transduced at a density of 1 × 106 cells/mL in the presence of LentiBOOST enhancer, and transduced cells (without sorting) were transplanted by retro-orbital injection into lethally irradiated (7 + 4 Gy, split dose) CD45.1 recipients (B6.SJL-Ptprca Pepcb/BoyJ, Jax Strain #002014) 24 h after transduction. PB samples were collected at weeks 4, 8, 12, and 16 to measure engraftment by flow cytometry (CD45.2/CD45.1), determine RBC indices, and quantitate sickled cells. At week 16, mice were euthanized, and BM cells were used to measure engraftment by flow cytometry (CD45.2/CD45.1), VCN, and mRNA expression, spleens were collected to weigh. All animal experiments were approved by the Boston Children's Hospital's Institutional Animal Care and Use Committee.
Stem cell growth medium
Manufactured by CellGenix
Sourced in Germany, United States
Stem Cell Growth Medium is a liquid cell culture medium specifically designed to support the in vitro growth and expansion of human stem cells. It provides a balanced and optimized combination of essential nutrients, growth factors, and other components required for maintaining the undifferentiated state and promoting proliferation of stem cells.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (2 mentions)
Cd3 positive selection kit, by STEMCELL (2 mentions)
Mouse lineage cell depletion kit, by Miltenyi Biotec (1 mentions)
Mouse scf, by Thermo Fisher Scientific (1 mentions)
Hflt3l, by Thermo Fisher Scientific (1 mentions)
Mouse il 3 mil 3, by Thermo Fisher Scientific (1 mentions)
Human il 15, by R&D Systems (1 mentions)
Easysep human nk cell isolation kit, by STEMCELL (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Perforin fitc, by Miltenyi Biotec (1 mentions)
Human male ab serum, by Merck Group (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cd56 fitc, by Miltenyi Biotec (1 mentions)
Cd3 apc, by Miltenyi Biotec (1 mentions)
Dynabeads cd3, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Miltenyi cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Fms like tyrosine kinase 3 ligand, by Thermo Fisher Scientific (1 mentions)
10 protocols using stem cell growth medium
1
Lentiviral gene therapy for sickle cell disease
2
Expansion of Human NK Cells
3
NK Cell-Mediated Cytotoxicity Assay
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K562mbIL15-41BBL feeder cell line (107 cells) was co-cultured with 1.5 × 107 PBMCs for 2 weeks in stem cell growth medium (Cellgenix), with 10% human male AB serum (Sigma) to obtain highly purified NK cells. IL-2 (10 IU/mL) was added for the first week and 100 IU/mL thereafter. NK cells were then exposed to both sorted GFP+ and CD14+GFP+ cellular entities. After cell permeabilization (Cytofix/Cytoperm kit, BD Biosciences), CD3−CD56+CD16+ NKs perforin generation was detected with specific Perforin-FITC (Miltenyi-130 096 668). Cytotoxicity was monitored with conventional 2 h Europium lysis-terpyridine dicarboxylic acid (TDA) release assay (Perkin-Elmer). NK (effector) cells loaded TDA-labeled (target) H460-CSC-GFP+ and CD14+GFP+ hybrid cells, at effector-to-target cell ratios of 50:1, 25:1, 12.5:1 and 6.25:1. Fluorescent measures (ex: 340 nm; em: 612 nm) were assessed in a BioTek Epoch Microplate Spectrophotometer.
4
Expanded Natural Killer Cell Generation
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Activated NK cells expansion is as described in previous studies [13 (link)]. In short, peripheral blood mononuclear cells (PBMC) were incubated with 100 Gy-irradiated K562-mbIL15-41BBL cells in Stem Cell Growth Medium (SCGM; CellGenix, Freiburg, Germany) supplemented with 10% FBS and 10 U/mL rIL-2. Depletion of CD3 positive T cells was performed after 1 week using CD3 DynaBeads (Invitrogen, Carlsbad, CA, USA). Purified NK cells were expanded further with rIL-2 supplemented SCGM for 1 more week. After the expansion, purity of expanded natural killer cells was assessed by flow cytometry. The cells were analyzed using FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). The purity of the expanded NK cells was confirmed using CD3-APC (Miltenyi, Singapore) and CD56-FITC (Miltenyi, Singapore) and the cell viability was more than 92%. The expanded NK cells used in this study contained less than 5% of CD3+CD56− T cells. The PBMC and the expanded NK cells derived from them were a kind gift from Dr. Dario Campana’s laboratory at Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore.
5
Transduction of SCD CD34+ HSPCs
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SCD patient CD34+ HSPCs were isolated from unmobilized PB following receiving Boston Children’s Hospital institutional review board approval and informed patient consent. The SCD CD34+ HSPCs were enriched using the Miltenyi CD34 Microbead kit (Miltenyi Biotec, Auburn, CA, USA). CD34+ cells were prestimulated for 36–40 h at 1 × 106 cells/mL in Stem Cell Growth Medium (CellGenix, Portsmouth, NH, USA) supplemented with SCF, FMS-like tyrosinekinase 3 ligand, and TPO, all from Peprotech. Cells were then enumerated and transduced with the vector at an MOI as indicated in presence of LentiBOOST enhancer (SIRION Biotech, Gräfelfing, Germany) for 24 h before downstream processing.
6
Expansion of Primary NK Cells
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Owing to the limited supply of patient NK cells, NK cells from XLP1 patients were expanded to study molecular signals for NF-κB activation and effector functions. NK cells from normal donors were also expanded and, after a period of rest, reproduced the synergistic increase in effector functions and signalling following stimulation with NKG2D and 2B4. Primary NK cells obtained from normal or XLP1 donors were expanded by stimulation with the K562-mb15-41BBL cell line, as previously described50 (link)51 (link) with slight modifications. 1.5 × 106 PBMCs were cultured with 1 × 106 100 Gy gamma ray-irradiated K562-mb15-41BBL feeder cells in stem cell growth medium (CellGenix) supplemented with 10% FBS and 10 U ml−1 rIL-2. The medium was changed every 2 days and replaced with fresh medium containing 10 U ml−1 rIL-2. After 1 week of coculture, residual T cells were depleted using the CD3+ selection kit (StemCell Technologies). The remaining cells were further expanded for an additional 2 weeks in stem cell growth medium supplemented with 10% FBS, 100 U ml−1 rIL-2 and 5 ng ml−1 rIL-15. The resulting cell population was 97–99% CD3-CD56+, as assessed using flow cytometry.
7
Expansion of Primary Human NK Cells
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Primary human NK cells were expanded as previously described [29 (link)] with some modifications. PBMCs (1.5 × 106) were incubated in a 24-well tissue culture plate with 100 Gy-irradiated K562-mb15-41BBL cells (1 × 106) in Stem Cell Growth Medium (SCGM; CellGenix, Freiburg, German) supplemented with 10% FBS and 10 U/mL rIL-2. The medium was exchanged every 2 days with fresh medium with rIL-2. After 1 week, residual T cells were depleted with a CD3 positive selection kit (STEMCELL Technologies, Cambridge, MA, USA). Purified NK cells were incubated in SCGM supplemented with 10% FBS, 100 U/mL rIL-2, and 5 ng/mL rIL-15 for 2 additional weeks with a medium exchange every 2 days. The expanded cell populations were 96–99% CD3−CD56+ as assessed by flow cytometry.
8
Protocols for Mammalian Cell Culture
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All mammalian cell cultures were performed in a 37°C incubator with 5% CO2. Lymphocyte media were composed of RPMI 1640 (HyClone, Logan, UT, USA) with 10% fetal calf serum (FCS; Omega Scientific, Tarzana, CA, USA), 10 mM HEPES (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), 55 μM 2-mercaptoethanol (Gibco), and 100 IU/mL penicillin-streptomycin (HyClone). Murine HSC media were either StemSpan serum-free expansion medium (SFEM) (STEMCELL Technologies, Vancouver, BC, Canada; Figures 1 and 2 ) or stem cell growth medium (SCGM) (CellGenix, Portsmouth, NH, USA; Figures 3 , 4 , 5 , and 6 ; Figure S10 ), with both having the following additives: 2% FCS, 1× GlutaMAX (Gibco), 100 IU/mL penicillin-streptomycin, and recombinant murine cytokines stem cell factor (SCF) (50 ng/mL) and thyroperoxidase (TPO) (20 ng/mL; both PeproTech, Cranbury, NJ, USA). Human HSC media were SCGM with 100 ng/mL each recombinant human TPO, SCF, and Flt3 (PeproTech).
9
Cell Culture Protocols for Hematological Malignancies
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TOM-1 (DMSZ, ACC 578) and MOLM-13 cells (DMSZ, ACC 554) were cultured in complete RPMI 1640 medium supplemented with 20% or 10% fetal calf serum (FCS), respectively (all purchased from Merck Millipore, Burlington, MA, USA). HEK293T (ATCC, CRL-11268), HT-1080 (ATCC, CCL-121), and OCI-AML3 cells (DMSZ, ACC 582) were cultured in DMEM, high glucose, GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS. Primary AML blasts and CD34+ HSPCs were cultured in stem cell growth medium (SCGM) (CellGenix, Portsmouth, NH, USA) supplemented with 1% human serum albumin (HSA) (Baxter, Deerfield, IL, USA), 10 ng/mL Flt3L, 10 ng/mL SCF, and 10 pg/mL IL-3 (all purchased from PeproTech, Rocky Hill, NJ, USA). ALL blasts were cultured in adapted Iscove’s modified Dulbecco’s medium (IMDM) medium59 (link) (Thermo Fisher Scientific, Waltham, MA, USA). Mycoplasma tested cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
10
Expansion of Primary Human NK Cells
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Primary human NK cells isolated from PBMCs were expanded as previously described [34 (link)], with some modifications. PBMCs (1.5 × 106 cells) were incubated in a 24-well tissue culture plate with 100 Gy-irradiated K562-mb15-41BBL cells (1 × 106 cells) in Stem Cell Growth medium (SCGM; CellGenix, Freiburg, Germany) supplemented with 10% FBS and 10 U/mL rIL-2. The half volume of medium was exchanged every 2 days with fresh medium containing 10 U/mL rIL-2. After a week, residual T cells were depleted with a CD3 positive selection kit (StemCell Technologies. The remaining cells were incubated in SCGM supplemented with 10% FBS, 100 U/mL rIL-2, and 5 ng/mL rIL-15 (PeproTech, Rocky Hill, NJ, USA) for 2 additional weeks, with a half-medium exchange every 2 days. NK cell populations in PBMCs expanded on average 2000 fold by a total of 3 weeks. The expanded cell populations were 96–99% CD3−CD56+, as assessed by flow cytometry.
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