Advanced DMEM, IMDM, RPMI-1640, l -glutamine, penicillin/streptomycin (P/S), and insulin-transferrin-selenium (ITS) were purchased from GIBCO/Invitrogen (Waltham, MA, USA). StemSpan serum-free expansion medium (SFEM) was purchased from STEMCELL Technologies (Vancouver, Canada). Phosphate-buffered saline was purchased from Merck Life Science SL (Darmstadt, Germany). Human (h) stem cell factor (SCF), hFMS-like tyrosine kinase 3 ligand (FLT3-L), hIL-3, hIL-7, and hIL-15 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD3 (OKT3) and anti-CD28 (CD28.2) mAbs, 7-amino-actinomycin D (7-AAD), and fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridin chlorophyll (PerCP)-, allophycocyanin (APC)-, phycoerythrin/cyanine7 (PE/Cy7)-, Brilliant Violet 421 (BV421)-, and BV510-conjugated mAbs specific for human CD3 (SK7), CD19 (HIB19), CD22 (HIB22), CD10 (HI10a), CD13 (WM15), CD45 (HI30), HLA-ABC (G46-2.6), CD25 (M-A251), CCR7 (150503), CD27 (L128), CD45RO (UCHL1), LAG3 (T47-530), TIM3 (7D3), PD-1 (MIH4), and isotype-matched negative control mAbs, were purchased from BD Biosciences (Franklin Lakes, NJ, USA), CD69 (REA824) from Miltenyi Biotec, and anti-His (J095G46) from BioLegend (San Diego, CA, USA).
Hil 3
Manufactured by Miltenyi Biotec
Sourced in Germany
The HIL-3 is a laboratory equipment designed for cell isolation and separation. It utilizes a technology that enables the efficient and gentle separation of cells based on their specific characteristics. The core function of the HIL-3 is to facilitate the isolation and purification of target cell populations from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
H tpo, by Miltenyi Biotec (4 mentions)
H flt3 ligand, by Miltenyi Biotec (3 mentions)
Hil 7, by Miltenyi Biotec (2 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (2 mentions)
Automacs pro, by Miltenyi Biotec (2 mentions)
X vivo, by Lonza (2 mentions)
Allophycocyanin, by BD (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Stemspan serum free expansion medium, by STEMCELL (1 mentions)
Hil 15, by Miltenyi Biotec (1 mentions)
Phycoerythrin pe, by BD (1 mentions)
Pd 1 mih4, by BD (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Advanced dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Eurobio Scientific (1 mentions)
Ficoll, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
6 protocols using hil 3
1
Expansion of Human Hematopoietic Cells
2
Isolation and Expansion of Human CD34+ Cells
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Mononuclear cells were isolated from umbilical cord blood from anonymous healthy donors by density centrifugation using Ficoll (Biocoll, Merck Millipore). Human CD34+ cells were then enriched in the sample by immunomagnetic beads using an AutoMACSpro (Miltenyi Biotec). After collection, enriched CD34+ cells were frozen in a cryopreservation medium containing 90% of fetal bovine serum (Eurobio) and 10% of dimethylsulfoxide (Sigma) and stored in liquid nitrogen.
After thawing, the CD34+ cells were cultured in a 96-well plate in a humidified 5% CO2 incubator at 37°C. Cells were cultured in prestimulation medium made of XVivo (Lonza) supplemented with penicillin/streptomycin (respectively, 100 U/mL and 100 μg/mL; Gibco, Thermo Fisher Scientific), 50 ng/ml h-FLT3-ligand, 25 ng/ml h-SCF, 25 ng/ml h-TPO, and 10 ng/ml h-IL3 (Miltenyi) final concentration.
After thawing, the CD34+ cells were cultured in a 96-well plate in a humidified 5% CO2 incubator at 37°C. Cells were cultured in prestimulation medium made of XVivo (Lonza) supplemented with penicillin/streptomycin (respectively, 100 U/mL and 100 μg/mL; Gibco, Thermo Fisher Scientific), 50 ng/ml h-FLT3-ligand, 25 ng/ml h-SCF, 25 ng/ml h-TPO, and 10 ng/ml h-IL3 (Miltenyi) final concentration.
3
Cryopreserved CD34+ Cell Expansion
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Human CD34+ cells were isolated from the mononuclear fraction of UCB samples using the autoMACSpro (Miltenyi Biotec, Paris, France) immunomagnetic cell separation system. They were then cryopreserved in Cryostor (StemCell, Paris, France) and stored in liquid nitrogen or used directly without freezing.
Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 in a 24-well plate in X-VIVO (Lonza) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco, Thermo Scientific), 50 ng/ml h-FLT3, 25 ng/ml h-SCF, 25 ng/ml h-TPO, and 10 ng/ml h-IL3 (Miltenyi Biotec, Paris, France) final concentration. VPA (Sigma Aldrich) was used at a final concentration of 1.25 mM.
Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 in a 24-well plate in X-VIVO (Lonza) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco, Thermo Scientific), 50 ng/ml h-FLT3, 25 ng/ml h-SCF, 25 ng/ml h-TPO, and 10 ng/ml h-IL3 (Miltenyi Biotec, Paris, France) final concentration. VPA (Sigma Aldrich) was used at a final concentration of 1.25 mM.
4
Isolation and Culture of AML Cells
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Peripheral blood or bone marrow samples derived from AML patients between 2018 and 2020 were obtained from the UCT Biobank of the University Hospital Frankfurt. The use of peripheral blood and bone marrow aspirates was approved by the Ethics Committee of Frankfurt University Hospital (approval no. SHN-03-2017). All patients gave informed consent to the collection of samples and to the scientific analysis of their data and of biomaterial obtained for diagnostic purposes according to the Declaration of Helsinki.
Mononuclear cell (MNC) fractions were purified by gradient centrifugation with Biocoll cell separation solution (Merck Millipore, Darmstadt, Germany). Leukemic cells were enriched by negative selection with a combination of CD3-, CD19- and CD235a-microbeads (all obtained from Miltenyi Biotec, Bergisch Gladbach, Germany, 130–050-301, 130–050-101, 130–050-501) according to the manufacturer’s instructions and separated by the autoMACS™ Pro Separator (Miltenyi Biotec). FACS staining and treatment for viability assays of AML blasts was executed immediately after isolation. Culture medium for AML blasts consisted of IMDM (Biochrom) supplemented with 10% FBS, 4 mM L-glutamine, 25 ng/ml hTPO, 50 ng/ml hSCF, 50 ng/ml hFlt3-Ligand and 20 ng/ml hIL-3 (all obtained from Miltenyi Biotec, 130–094-013, 130–096-695, 130–096-479, 130–095-069).
Mononuclear cell (MNC) fractions were purified by gradient centrifugation with Biocoll cell separation solution (Merck Millipore, Darmstadt, Germany). Leukemic cells were enriched by negative selection with a combination of CD3-, CD19- and CD235a-microbeads (all obtained from Miltenyi Biotec, Bergisch Gladbach, Germany, 130–050-301, 130–050-101, 130–050-501) according to the manufacturer’s instructions and separated by the autoMACS™ Pro Separator (Miltenyi Biotec). FACS staining and treatment for viability assays of AML blasts was executed immediately after isolation. Culture medium for AML blasts consisted of IMDM (Biochrom) supplemented with 10% FBS, 4 mM L-glutamine, 25 ng/ml hTPO, 50 ng/ml hSCF, 50 ng/ml hFlt3-Ligand and 20 ng/ml hIL-3 (all obtained from Miltenyi Biotec, 130–094-013, 130–096-695, 130–096-479, 130–095-069).
5
CAR T Cell Cytotoxicity Assay for B-ALL
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Target cells (cell lines or primary B-ALL cells; 100,000 target cells/well of a 96-well plate) were incubated with CD22-CAR or mock-IC T cells at different effector:target (E:T) ratios for the indicated time periods. Cell lines were cultured in RPMI-1640, 10% FBS and penicillin–streptomycin. Primary cells were cultured in StemSpanTM SFEM media (StemCell Technologies, Vancouver, Canada), 20% FBS, penicillin–streptomycin, insulin–transferrin–selenium (Gibco/Invitrogen), hSCF (100 ng/mL), hFLT3L (100 ng/mL), hIL3 (10 ng/mL) and hIL7 (10 ng/mL, all from Miltenyi Biotec, Bergisch Gladbach, Germany). CAR T cell-mediated cytotoxicity was determined by analyzing the residual live (7-aminoactinomycin D–; 7-AAD–) target cells at each time point and E:T ratio. For absolute cell counting, BD TruCountTM absolute count tubes (BD Biosciences) were used. Quantification of the proinflammatory cytokines IL2, TNFα and IFNγ was measured by ELISA using BD OptEIATM Human ELISA kits (BD Biosciences), in supernatants harvested at 24 hours (cell lines) and 48 hours (primary cells) post T cell-exposure, using an 1:1 E:T ratio. Online supplementary table 1 shows clinic-biological features of the B-ALL samples used.
6
Primary AML Sample Cultivation
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Primary human AML samples were obtained with informed consent and used in agreement with the Declaration of Helsinki upon the approval of the local ethic committees of the Goethe University Frankfurt (approval number 329–10). Samples were maintained in X-Vivo10 medium (Lonza) supplemented with 10% FCS (Hyclone/Perbio Science), with the addition of 20 ng/ml hIL-3, 50 ng/ml hSCF, 25 ng/ml hTPO and 50 ng/ml hFLT3-ligand (Miltenyi, Bergisch-Gladbach, Germany).
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