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Macsductin™ Reagent Macsductin™ 1.2 Page 1/4 MACSductin™ Reagent 0.25 mL 130-097-256 0.5 mL 130-097-257 3×0.5 mL 130-097-259 Contents magselectofection, superior viral transduction efficiencies of both primary murine and human target cells were demonstrated compared 1. Description to standard transduction methods or Magnetofection alone.³ 1.1 Combined magnetic cell sorting and viral transduction Magnetic labeling of both virus particles and the target cells ensures 1.2 Product applications an efficient colocalisation within the high-gradient magnetic field of 1.3 Reagent and instrument requirements the MACS Column leading to reduced viral reagent requirements 2. Protocol and specific transduction of only the magnetically labeled target cells. 2.1 Calculation of reagent requirements 2.2 Transduction of positively selected target cells Magnetically label virus with 2.3 Transduction of cell monocultures MACSductin Reagent. 3. Examples of magnetic transduction using MACSductin™ Reagent Mix MACSductin labeled virus 3.1 Transduction of human cell lines with adenoviral vector with magnetically labeled cells. 4. Optimization of transduction parameters 5. Troubleshooting 6. References Apply labeled virus and cells to column. 1. Description Components 0.25 mL MACSductin™ Reagent (# 130-097-256) or 0.5 mL MACSductin Reagent (# 130-097-257) or 3×0.5 mL MACSductin Reagent (# 130-097-259) Elute virus-cell complexes in cell Size 0.25 mL for transduction of 10⁷–5×10⁸ cells or culture medium and culture cells. 0.5 mL for transduction of 2×10⁷–10⁹ cells or 3×0.5 mL for transduction of 6×10⁷–3×10⁹ cells Product format MACSductin Reagent is supplied in a sterile solution without preservatives. Storage Store protected from light at 2 − 8 °C. Do not freeze. The expiration date is indicated on the vial label. 1.1 Combined magnetic cell sorting and viral transduction During the infection of target cells with native or recombinant viruses in cell culture, the major factor limiting infection is the diffusion of the virus particles to the cell surface.¹ As the half life of viruses in cell culture is short (e.g. 5–8 hours for retroviruses)¹, it is important to ensure an efficient and rapid delivery of the virus to the target cells to maximize infection or transduction events. Legend MACSductin Reagent combines two established technologies MACSductin Reagent magnetically labeled cell into a single process to significantly improve viral transduction efficiencies and improve sample handling: MACS® Technology virus and Magnetofection®. MACS Technology is the gold standard in cell separation. Target cells are specifically labeled with antibody- Figure 1: Principle of magnetic transduction with MACS Technology. conjugated superparamagnetic nanoparticles, MACS MicroBeads, approximately 50 nanometers in diameter. Labeled cells are then specifically retained within a MACS Column when it is placed in a 1.2 Product applications MACS Separator. This is a strong permanent magnet which induces a ● high-gradient magnetic field on the column matrix. Magnetofection Ex vivo genetic modification of primary human and murine cell subpopulations. 140-003-567.01 is a method that enables cell modifying agents (e.g. RNA, DNA, viruses) to be concentrated by magnetic force at the target cell’s ● Effective transduction of target cells with low titer virus surface using magnetic polycationic carriers.² preparations – no viral concentration required. When the two methods are combined into a single process, termed ● Synchronized infection of target cells. Miltenyi Biotec GmbH Miltenyi Biotec Inc. Friedrich-Ebert-Straße 68, 51429 Bergisch Gladbach, 2303 Lindbergh Street, Auburn, CA 95602, USA Germany Phone 800 FOR MACS, +1 530 888 8871, Fax +1 530 888 8925 Phone +49 2204 8306-0, Fax +49 2204 85197 [email protected] [email protected] www.miltenyibiotec.com page 1/4 ● Effective transduction of difficult-to-transduce target cells, e.g., Reagent will complex both infectious and defective virus particles, leukaemia cell lines. the infectious titer of the virus sample will only allow an approximate ● Compatible with a wide range of recombinant and wild-type estimation of the total virus particle titer to be calculated. As a viruses. general rule of thumb, 1 in 1000 retro-/lentivirus vector particles in unpurified supernatant are infectious while 1 in 100 retro-/lentivirus 1.3 Reagent and instrument requirements vector particles in purified vector preparations are infectious. Generally, for purified adenoviral vectors more than 1 in 100 particles ● Recombinant virus containing expression cassette of interest. are infectious (e.g. 1 in 20) and the physical particle number is often ● Magnetically labeled target cell population enriched by MACS used as a measurement of viral titer. Technology. For example, for a standard lentiviral transduction of 1×10⁶ target ● PEB buffer (degassed): Prepare a solution containing phosphate- cells at a multiplicity of infection of 1 (MOI=1) with unpurified buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin supernatant, 25 µL of MACSductin Reagent is required. When using (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution purified lentiviral particles, the MACSductin Reagent requirement (# 130-091-376) 1:20 with autoMACS® Rinsing Solution can be reduced by a factor of 10 to 2.5 µL. Some further examples (# 130-091-222). Keep buffer cold (2−8 °C). of how to calculate reagent amounts for a lentiviral transduction are ▲ Note: EDTA can be replaced by other supplements such as anticoagulant shown in table 1. citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, Target MOI No. of MACSductin Minimum MACS or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not cell infectious Reagent (µL) complex Column recommended for use. number virions (IU) volume (µL) ● MACS Columns and MACS Separators: Choose the appropriate Unpurified supernatant MACS Separator and MACS Columns according to the number of 1×10⁶ 0.1 1×10⁵ 2.5 10 MS labeled cells that are to be transduced and to the number of total cells. 1×10⁶ 1 1×10⁶ 25 75 MS Column Min. number Max. number Separator of labeled of labeled 5×10⁶ 0.1 5×10⁵ 12.5 37.5 LS cells to be cells to be transduced transduced 1×10⁷ 0.5 5×10⁶ 125 375 LS MS 10⁶ 5×10⁶ MiniMACS, OctoMACS, Purified vector preparation VarioMACS, SuperMACS II 1×10⁶ 0.1 1×10⁵ 0.25 10 MS LS 2.5×10⁶ 10⁷ MidiMACS, QuadroMACS, 1×10⁶ 1 1×10⁶ 2.5 10 MS VarioMACS, SuperMACS II 5×10⁶ 0.1 5×10⁵ 1.25 10 LS ▲ Note: For very rare cells, such as CD34+ progenitor cells, 10⁵ cells can also be efficiently transduced in an MS Column when using an MOI≥10. 1×10⁷ 0.5 5×10⁶ 12.5 37.5 LS ▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective Table 1: Examples of MACSductin Reagent requirements for lentiviral vector MACS Separator data sheet. transduction using unpurified supernatant and purified vector preparation. ● MACS MultiStand (# 130-042-303) ▲ To accurately determine the absolute physical particle titer of the virus samples, it is recommended to use an appropriate virus particle ● MS Columns (# 130-042-201) or LS Columns (# 130-042-401) titering kit (e.g. virus-associated p24 ELISA or (RT-) PCR based for ● Cell culture medium, e.g. RPMI 1640 (# 130-091-440) and lentiviral vectors) or a DNA slot-blot approach⁴ (for adenoviral vectors). required supplements ▲ The virus aliquot should have a volume at least twice the volume ● 1.5 mL, 15 mL, and 50 mL sterile tubes of MACSductin Reagent. If required, dilute virus aliquot with serum- free medium before use. 2. Protocol ▲ For optimum performance, the minimum complex-forming Before starting volume used should not be less than 10 µL and maximal complex volumes should not exceed 2 mL for transduction using an MS ▲ The target cell population must be magnetically labeled with Column or 5 mL for transduction using an LS Column. MACS MicroBeads. These cells can either comprise the enriched fraction from a positive selection procedure, or they can be a cell 2.2 Transduction of positively selected target cells monoculture that have been uniformly magnetically labeled with MicroBeads. 1. Thaw virus sample. Adenoviruses should be thawed on ice, ▲ It is recommended to pre-enrich the target cells from mixed cell retro- and lentiviral samples should be thawed rapidly at populations prior to transduction as the presence of the MACSductin 37 °C. If the virus supernatant has not been previously purified, Reagent within the MACS Separation Columns can reduce cell briefly centrifuge sample at 13,000×g for 30 seconds to remove selection efficiency. particulate matter. Transfer supernatant to a fresh tube, avoiding floating fragments. Alternatively, filter through an appropriate ▲ Before transducing cell monocultures, it is recommended to assess sterile filter (0.22 or 0.45 µm). the presence of a suitable cell surface molecule for magnetic labeling by flow cytometry. For an overview of available reagents for use in the 2. Following positive selection of magnetically labeled target cells field of hematology and immunology refer to www.miltenyibiotec.com. (see “Before starting” above), determine the target cell number in the positive fraction. 2.1 Calculation of reagent requirements 140-002-817.01 3. Calculate amount of virus sample required to transduce target The efficient formation of magnetic virus transduction complexes is cells at desired MOI. dependent on the ratio of MACSductin Reagent to virus particles. As not all virions in a virus sample are infectious and MACSductin Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use. page 2/4 4. Resuspend MACSductin Reagent by pipetting or vortexing.
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