Heparinized bone marrow samples were collected from 6 patients with newly diagnosed Ph+ B-ALL (detailed information for these patients are provided in Supplementary Table S2 ). Mononuclear cells (MNCs) were then separated by density gradient centrifugation using Lymphoprep reagent (Stemcell Technologies). Subsequently, MNCs were cultured in StemSpan basic media (Stemcell Technologies) supplemented with 10 ng/mL human stem cell factor, 10 ng/mL human IL-3, 10 ng/mL human IL-6 (all above cytokines were purchased from R&D Systems), 100 U/mL penicillin, and 100 μg/mL streptomycin (both from BBI Life Sciences). This study was approved by the Institutional Review Board of the Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Informed consent for the in vitro drug testing studies was obtained in accordance with the Declaration of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine.
Human il 3
Manufactured by R&D Systems
Sourced in Israel, China
Human IL-3 is a recombinant human cytokine that functions to stimulate the growth and differentiation of hematopoietic progenitor cells. It is a glycoprotein and a member of the interleukin family.
Automatically generated - may contain errors
Lab products found in correlation
Human il 6, by R&D Systems (4 mentions)
Human scf, by R&D Systems (3 mentions)
Human stem cell factor, by R&D Systems (2 mentions)
Lymphoprep reagent, by STEMCELL (2 mentions)
Penicillin, by BBI Lifesciences (2 mentions)
Streptomycin, by BBI Lifesciences (2 mentions)
Erythropoietin, by Amgen (2 mentions)
Recombinant human insulin, by Merck Group (2 mentions)
Heparin, by Merck Group (2 mentions)
Human holo transferrin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Hydrocortisone sodium hemisuccinate, by Merck Group (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Methocult, by STEMCELL (1 mentions)
Human scf, by ProSpec (1 mentions)
Human g csf, by ProSpec (1 mentions)
Stemspan sfem medium, by STEMCELL (1 mentions)
Ifn β, by BioLegend (1 mentions)
7 protocols using human il 3
1
Isolation and Culture of Ph+ B-ALL Cells
2
Hematopoietic Stem Cell Enrichment and Culture
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CD34+ cells were purified from mononuclear cells of UCB by using immunomagnetic cell separation (Dynabeads; Invitrogen, https://www.thermofisher.com ). Cells were cultured in DMEM supplemented with each batches of fetal bovine serum (FBS) in the presence of 100ng/ml human Flt-3 ligand (Prospec Tany, Rehovot, Israel), 100ng/ml human SCF (Prospec Tany), 40ng/ml human IL-6 (R&D Systems), 40ng/ml human IL-3 (R&D Systems) and 40ng/ml human G-CSF (Prospec Tany) supplemented with 10−6 M hydrocortisone sodium hemisuccinate (Sigma). For co-culture, MSCs were irradiated (1500 cGy) before use and co-cultured with CD34+ cells in similar medium conditions for 5 days as described [13 (link)]. For colony forming assay of hematopoietic progenitors, hematopoietic cells were plated for 14 days in semi-solid methylcellulose media (MethoCult; StemCell Technologies) containing cytokines, and analyzed for colony numbers and lineages as described[13 (link)]. For long-term culture-initiating cell (LTC-IC) analysis, CD34+ cells were co-cultured with normal MSCs for 5 days, transferred to a 6-week long-term culture, and subjected to a colony-forming assay in semi-solid medium.
3
In Vitro Erythroid Differentiation from CD34+ HSPCs
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Primary human CD34+ HSPCs from de-identified, healthy adult donors following G-CSF mobilization were acquired from the Excellence in Molecular Hematology at the Fred Hutchinson Cancer Research Center (Seattle, Washington). CD34+ HSPCs were subjected to erythroid differentiation conditions using a three phase culture system as previously described3 (link),53 (link). Erythroid differentiation medium (EDM) was created as follows: IMDM (CellGro) supplemented with 330 µg/mL holo-human transferrin (Sigma), 10 µg/mL recombinant human insulin (Sigma), 2 IU/mL heparin (Sigma), 5% human solvent detergent pooled plasma AB (Rhode Island Blood Center), 3 IU/mL erythropoietin (Amgen), 1% L-glutamine (Life Technologies), and 2% penicillin/streptomycin (Life Technologies). Phase I medium consisted of EDM supplemented with 10−6 M hydrocortisone (Sigma), 100 ng/mL human SCF (R&D), and human IL-3 (R&D). Phase II medium consisted of EDM supplemented with 100 ng/mL SCF. Phase III medium consisted of EDM without additional supplementation. CD34+ HSPCs were thawed into Phase I medium and were maintained in this medium for the first 7 days of culture. Cells were switched to Phase II medium for days 7–11 of culture. Cells were switched to Phase III medium for days 11–18 of culture.
4
Isolation and Culture of AML Mononuclear Cells
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Bone marrow (BM) samples were collected from patients with AML (patient details are provided in Supplementary Table S1). Mononuclear cells were separated with Lymphoprep reagent (Stemcell Technologies) using density gradient centrifugation. They were subsequently cultured in StemSpan SFEM medium (Stemcell Technologies) supplemented with 10 ng/mL human stem cell factor, 10 ng/mL human IL3, 10 ng/mL human IL6 (all cytokines were purchased from R&D Systems), 100 U/mL penicillin, and 100 μg/mL streptomycin (both from BBI Life Sciences).
Written informed consents were obtained from all patients in accordance with the Declaration of Helsinki, and all manipulations were approved by the Institutional Review Board of Guangzhou First People's Hospital, School of Medicine, South China University of Technology (Guangzhou, P.R. China).
Written informed consents were obtained from all patients in accordance with the Declaration of Helsinki, and all manipulations were approved by the Institutional Review Board of Guangzhou First People's Hospital, School of Medicine, South China University of Technology (Guangzhou, P.R. China).
5
In Vitro Erythroid Differentiation from CD34+ HSPCs
6
Cytokine Quantification by ELISA
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Mouse: Secreted TNFα (Biolegend, San Diego, CA, USA), IL-10 (Biolegend), IL-1β (Biolegend), IFNλ (R&D systems), IFNβ (Biolegend) and IFNα (R&D systems) were measured by ELISA according to the manufacturer’s instructions. Human: Secreted IFNλ (R&D Systems), IFNγ (Biolegend), IL-10 (Biolegend) and TNFα (Biolegend) were measured by enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Quantification of human IL-3 (R&D Systems) was performed in combination with chemiluminescent detection (R&D Systems) for increased sensitivity. The assays were performed according to the manufacturer’s instructions and measured in a microplate reader set to luminescence mode (BMG Labtech, Ortenberg, Germany) with an integration time of 2 seconds per well, yielding a sensitivity of 3.9 pg/ml IL-3.
7
Maintenance and Differentiation of Stem Cells
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HDFs and iMEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS, Japan Bio Serum). PBMCs were cultured in StemSpan ACF (STEMCELL) with 100 ng/mL human SCF (R&D), 100 ng/mL human TPO (R&D), 100 ng/mL human Flt3/Flk2 (R&D), 50 ng/mL human IL-6 (R&D), and 20 ng/mL human IL-3 (R&D) for 5 days before the SeV vectors infection. Primed PSCs were maintained in StemFit AK02N medium (Ajinomoto) on laminin 511-E8 fragments (iMatrix-511, Nippi)-coated plates. Naive PSCs were cultured on iMEFs and maintained in t2iLGö (Takashima et al., 2014 (link)) medium composed of N2B27 medium (NDiff227, Takara Bio) with 1 μM CHIR99021 (Merck), 1 μM PD0325901 (Merck), 10 μg/mL human LIF (PeproTech), and 2.5 μM Gö6983 (Merck). The day before naive PSCs were plated, iMEF cells were seeded in cell culture dishes at a concentration of 25,000 cells/cm2 and cultured overnight. The next day, the cells were washed twice with PBS(−) before plating. Medium was changed every other day and 10 μM Y27632 (Wako) was added just before every medium change. Primed iPSCs were passaged every 6–7 days, and naive iPSCs were every 3–4 days using Accutase (Innovative Cell Technologies).
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