The study was approved by the ethics evaluation committee of INSERM, institutional review board (IRB00003888) of the French institute of medical research and health (IORG0003254 FWA00005831). CB cells were obtained from Saint-Louis Hospital (Paris, France). mPB cells were collected from the residual cells in bags prepared for transplantation at Pitié Salpêtrière Hospital (Paris, France). Adult bone marrow cells were obtained from hip operations at the “Centre Hospitalier Intercommunal” (Créteil, France). The mononuclear cell fractions were isolated by gradient separation, and human CD34+ cells were isolated with the CD34+ progenitor cell isolation kit (Miltenyi Biotech, Paris, France) and the autoMACSPro instrument (Miltenyi Biotec).
Automacspro instrument
Manufactured by Miltenyi Biotec
Sourced in France, Germany
The AutoMACSPro is a fully automated magnetic cell separation instrument designed for the isolation of target cells from complex samples. It utilizes magnetic labeling of cells and magnetic separation to efficiently and reproducibly enrich target cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 progenitor cell isolation kit, by Miltenyi Biotec (1 mentions)
Mouse lineage cell depletion kit, by Miltenyi Biotec (1 mentions)
Methocult gf m3434 methylcellulose medium, by STEMCELL (1 mentions)
Whole blood cd45 microbeads, by Miltenyi Biotec (1 mentions)
Nucleospin blood kit, by Macherey-Nagel (1 mentions)
4 protocols using automacspro instrument
1
Isolation of Human CD34+ Cells
2
Isolation and Analysis of Murine Hematopoietic Cells
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Bone marrow cells were isolated by flushing in cold MACS buffer (phosphate-buffered saline with 0.5% bovine serum albumin and 2 mM EDTA pH8.0). The spleen and thymus were crushed through a 70-µm nylon mesh in a cold buffer. Following red blood cell lysis, cells were counted and resuspended at the appropriate dilution for further processing. Colony-forming assays were performed in MethoCult GF M3434 methylcellulose medium (STEMCELL Technologies) following the manufacturer’s protocol. Lineage-negative cells were enriched using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec #130-090-858), according to the manufacturer’s instructions and using an AutoMACS Pro instrument (Miltenyi Biotec). For CYTOF, lineage-negative cells were stained with metal-tagged antibodies and 500 nM IdU to label newly synthesized DNA as previously described (91 (link)). All antibodies and staining conditions are described in SI Appendix, Supplementary Methods .
3
Untreated Lung Cancer Patient Blood Analysis
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The study was approved by the Beijing Chest Hospital Committee on the Use of Humans as Experimental Subjects (Beijing China). In addition, all participants provided written informed consent. Patients were diagnosed with biopsy-validated lung cancer in clinical phase III or IV. The 57 male and 34 female patients had an age range of 32 to 87 years and a mean age of 62.3 years. Blood samples (4.5 ml) were collected into Vacuette EDTA anticoagulant tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) from 91 untreated patients with lung cancer at the Beijing Chest Hospital, and processed within 2 h using an autoMACS Pro instrument (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
4
Enrichment and VCN Determination of Human Cells in NSG Mice
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Human cells from NSG mouse bone marrow were enriched to facilitate VCN determination in these cells and to confirm the absence of human cells in immunodeficient mice as follows: single-cell suspensions of bone marrow cells (20–70 million cells) were labeled by incubation with 0.2 μL of whole-blood CD45 microbeads (Miltenyi Biotech) in 50 μL of PBS supplemented with bovine serum albumin (0.5%) and EDTA (2 mM) for 15 minutes, washed, resuspended in 1 mL, and sorted with an autoMACSPro instrument (Miltenyi Biotech) and the possel_s program. Sorted cells were then resuspended in PBS for the evaluation of cell enrichment by FACS and DNA extraction with the NucleoSpin Blood kit (Macherey Nagel). Relative enrichment varied between 1- and 20-fold, depending on the initial proportion of human cells (Figure S6 ). In mice with levels of hCD45+ cells below background levels (0.01%), no enrichment was observed, confirming the absence of human cells. Genomic DNA was extracted with the NucleoSpin Blood kit (Macherey Nagel, Hoerdt, France). Real-time PCR was performed with specific primers and probes (Supplemental Information ) using the 7300 ABI Prism Detection system and a 2× qPCR MasterMix containing Rox (Erogentec, Liege, Belgium). VCNs were determined from the results obtained with genomic DNA from a human cell line containing one copy of the integrated LV per haploid genome.
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