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As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is committed to providing our customers around the world with the highest quality products. In addition to direct selling in more than 20 countries in North America, Europe and Asia/Pacific, Miltenyi Biotec also provides support for our customers through an extensive distributor network covering dozens of additional countries.
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In this application protocol, we describe the viral transduction of human HSCs isolated from various sources, their concomitant expansion in defined cell culture conditions, and analysis of their function using flow cytometry or colony-forming assays.
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Prepare a solution with phosphate buffered saline (PBS), pH 7.2, and 2mM EDTA. Keep buffer cold (2–8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution. Keep buffer cold (2−8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as respective serum albumin, respective serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+
are not recommended for use.
Isolate mononuclear cells by density gradient centrifugation using Ficoll-Plaque™. Follow the Ficoll-Plaque™ protocol from the data sheet corresponding to the source tissue.
Download data sheet
Isolation of mononuclear cells from human peripheral blood
Isolation of mononuclear cells from bone marrow aspirates
Isolation of mononuclear cells from cord blood
Isolate CD34+ cells among the mononuclear cells using the CD34 MicroBead Kit UltraPure, human and LS or MS columns. Follow the protocol of the kit data sheet.
We recommend filtering the magnetically labeled cell suspension before separation on the column to guarantee a single-cell suspension.
Download kit data sheet
Isolated CD34+ cells can be transduced using retroviral or lentiviral vectors. Perform transduction either directly after enrichment or after pre-stimulation culture in StemMACS™ HSC Expansion Media XF supplemented with StemMACS HSC Expansion Cocktail.
Use Vectofusion-1®, a novel transduction enhancer, to boost transduction efficiency. Follow the protocol of the data sheet.
Download data sheet
For expansion, culture the isolated CD34+ cells in StemMACS™ HSC Expansion Media XF supplemented with StemMACS HSC Expansion Cocktail. Follow the protocol of the media data sheet.
Download data sheet
Analyze the expanded CD34+ cells for HSC-specific surface markers via flow cytometry using REAfinity™ antibodies, or perform CFU-GEMM analysis to assess their myeloid differentiation potential.
The following is a list of relevant resources that might be of interest:
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec B.V. & Co. KG and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec B.V. & Co. KG and/or its affiliates. All rights reserved.
Column | Max. number of labeled cells | Max. number of total cells | Separator |
---|---|---|---|
Positive or negative selection | |||
MS | 1×107 | 2×10⁸ | MiniMACS™, OctoMACS™, VarioMACS, SuperMACS II |
LS | 1×108 | 2×109 | MidiMACS™, QuadroMACS™, VarioMACS, SuperMACS II |
XS | 1×109 | 2×1010 | SuperMACS II |
Positive or negative selection | |||
autoMACS | 2×108 | 4×109 | autoMACS Pro, autoMACS |
Copyright © 2021 Miltenyi Biotec and/or its affiliates. All rights reserved.