T cells were obtained from buffy coats by Ficoll and magnetic T cell depletion (Miltenyi Biotec). T cells were expanded in Click’s media (50% RPMI, 50% Click’s (Irvine Scientific), 5% human serum, 1% Pen/Strep), activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) and IL-2 (100 IU/mL) every other day. Experiments were performed after 8–10 days of T cell expansion. Viral transduction was performed after 48 hours of the expansion at a MOI of 10. NK cells were selected from CB units by Ficoll and magnetic NK cell depletion (Miltenyi Biotec). To produce CAR-NK (ARI3 cells), NK cells from a CB unit were expanded with NK MACS Medium (Miltenyi Biotec), 1% NK MACS Supplement, 5% human serum, IL-2 (500 IU/mL) and IL-15 (140 IU/mL). On day 5, CB-NK were transduced at a multiplicity of infection (MOI) of 5 with Vectofusin-1 (10 mg/mL) following Vectofusin-1 protocol (Miltenyi Biotec). Cells were washed 6–24 hours after transduction. On day 7, ARI3 cells were coexpanded with feeder cells adding IL2 (400 IU/mL) every other day for the next 7 days. Of note, throughout the manuscript, ‘ARI3 cells’ refers to CAR-NK cells and CB-NK refers to untransduced NK cells.
Vectofusin 1
Manufactured by Miltenyi Biotec
Sourced in Germany
Vectofusin-1 is a cationic polymer-based transduction enhancer. It is designed to increase the efficiency of gene delivery in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Il 15, by Miltenyi Biotec (2 mentions)
Nk macs medium, by Miltenyi Biotec (2 mentions)
Atorvastatin, by Cayman Chemical (1 mentions)
Fluvastatin, by Cayman Chemical (1 mentions)
Rosuvastatin, by Cayman Chemical (1 mentions)
Pravastatin, by Cayman Chemical (1 mentions)
Simvastatin, by Cayman Chemical (1 mentions)
Prostaglandin e2 pge2, by Merck Group (1 mentions)
Il 21, by Merck Group (1 mentions)
Ggpp ammonium salt, by Merck Group (1 mentions)
Protamine sulfate, by Merck Group (1 mentions)
Dextran, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Vitamin c, by Merck Group (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Il 15, by CellGenix (1 mentions)
Il 1β, by Miltenyi Biotec (1 mentions)
Pen strep, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Human serum, by Thermo Fisher Scientific (1 mentions)
10 protocols using vectofusin 1
1
Expansion and Transduction of T Cells and CAR-NK Cells
2
Enhancing NK-92 Cell Cytotoxicity with Statins and Supplements
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NK-92 cells were seeded at 0.1 × 106 cells/mL in round-bottom 96-well plates (3799, Corning Life Sciences B.V., Amsterdam, the Netherlands). Atorvastatin (10493, Cayman Chemical), fluvastatin (10010337, Cayman Chemical), pravastatin (10010342, Cayman Chemical), rosuvastatin (12029, Cayman Chemical), and simvastatin (10010344, Cayman Chemical) were dissolved in DMSO (Sigma-Aldrich). Statins were used at final concentration of 20 μM, 5 μM, and 0.5 μM. DMSO was diluted in the same volume and served as solvent control. IL-2, IL-21 (Thermo Fisher Scientific), vitamin C (Sigma-Aldrich), dextran (Sigma-Aldrich), prostaglandin E2 (PGE2, Sigma-Aldrich), protamine sulfate (Sigma-Aldrich), and vectofusin-1 (Miltenyi Biotec) were dissolved in distilled water (GIBCO). DMSO was added 0.8 μL, 0.2 μL, and 0.02 μL, respectively, at the same volume as the statin group in 96-well plates. IL-21 was added at 20 ng/mL, 5 ng/mL, and 0.5 ng/mL. vitamin C was used at concentrations of 500 μg/mL, 50 μg/mL, and 5 μg/mL as described previously.23 (link) dextran was used at 80 μg/mL, 8 μg/mL, and 0.8 μg/mL.15 PGE2 was used at 100 μM, 10 μM, and 1 μM.22 (link) vectofusin-1 was used at 50 μg/mL, 5 μg/mL, and 0.5 μg/mL.14 (link),61 (link) GGPP ammonium salt was purchased from Sigma-Aldrich and was added at 10 μM in co-culture assays.29 (link)
3
Lentiviral Transduction of NK Cells
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Transductions were performed 7 days after the preparation of CD56+ CD3- cells and start of the NK cell expansion protocol. Briefly, lentiviral particles corresponding to an MOI of 1 (titered on K562/SKM1) were adjusted to a volume of 100 µl using plain NK MACS medium without additives, mixed with the equal volume of plain NK MACS supplemented with 5 µg/ml Vectofusin-1 (Miltenyi Biotec), incubated at room temperature for 8 min and mixed with 50 µl cell suspension containing 1 × 106 NK cells in NK MACS complete medium. For simultaneous double-transductions, both particle populations were used at MOIs of 1 and pooled prior to mixing with NK MACS and Vectofusin-1. Subsequently, the 250-µl cell/particle mix was transferred into 48-well plates and centrifuged for 90 min at 400g, 32°C. After spinoculation, cells were incubated at 37°C for additional 4 h before 500 µl NK MACS complete medium where carefully added to the cells.
4
Transduction of Cells with Lentiviral Vectors
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Parental HT1080, HT1080αHis, and HT1080CD62L cells were seeded at 8x103 cells per 96-well and incubated with serial dilutions of vector stocks. Transgene expression was analyzed 72 to 96 hours later by flow cytometry. Activated PBMC were seeded at 4x104 or 8x104 cells per 96-well, respectively, in TCM medium before CD62L-LV (5 µL or 10 µL) or VSV-LV (0.05 µL or 0.5 µL) were added. Where indicated, CD62L-LV transduction of PBMC was carried out in the presence of Vectofusin-1 (Miltenyi Biotec, Germany) as described previously (21 (link)). Cells were centrifuged at 850g and 32°C for 90 minutes, followed by the addition of TCM supplemented with cytokines. The medium was replenished every 2 to 3 days. Optionally, cells were passaged. Transgene expression was assessed by flow cytometry.
5
Lentiviral Transduction of NK-92 Cells
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NK-92 cells were transduced with lentiviral particles using spinoculation method. Briefly, low-volume NK-92 cells at the density of 5 × 105 cells/200 μL were incubated with the concentrated viral particles in the presence of Vectofusin-1 (10 μg/mL) (Miltenyi Biotec), followed by a spinoculation at 400 × g for 2 h at room temperature. After which, media containing Vectofusin-1 were added and incubated for additional 24 h before they were removed and replaced with fresh medium. NK-92 cells were evaluated for CAR expression by Fab detection as described above at 72 h posttransduction, and F(ab′)2-FITC-positive cells were enriched by fluorescence-activated cell sorting (FACS) using FACSAria cell sorter (BD Biosciences). Cell transduction and sorting were repeated thrice to ensure that more than 80% of the cells in culture were positive for F(ab′)2-FITC, an indication of anti-CD19 or anti-CD138 scFv expression. The enriched anti-CD19 CAR-NK-92 and anti-CD138 CAR-NK-92 cells, designated as CD19-NK-92 and CD138-NK-92 cells, respectively, were reevaluated for CD19-CAR or CD138-CAR expression by target antigens.
6
Lentiviral Transduction of Isolated NK Cells
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On day two post NK cell isolation NK cells were transduced with lentiviral particles.
Therefore, 0.5 × 106 cells per well were seeded in a flat bottomed 48-well plate. Subsequently, lentiviral particles and Vectofusin-1 (Miltenyi Biotec; 2.5 µg/ml final concentration per well) were mixed in identical volumes, incubated at room temperature for 7 min, and added to the cells, to reach a final cell concentration of 1 × 106 cells/ml. All previous steps were performed in serum-free NK-MACS® medium supplemented with 1% NK-MACS® Supplements, with or without 1% Pen/Strep and 80 ng/ml IL-1β (Miltenyi Biotec), 500 IU/ml IL-2 (Miltenyi Biotec or Novartis). Finally, 10 ng/ml IL-15 (Miltenyi Biotec or Peprotech) or 50 ng/ml IL-15 (CellGenix) was added. Subsequently, the plate was centrifuged at 400 × g for 2 h at 32 °C. Twenty-four hours post-transduction half of the medium was replaced by fresh medium containing 5% human plasma and a combination of IL-2 and IL-15 [24 (link)].
Therefore, 0.5 × 106 cells per well were seeded in a flat bottomed 48-well plate. Subsequently, lentiviral particles and Vectofusin-1 (Miltenyi Biotec; 2.5 µg/ml final concentration per well) were mixed in identical volumes, incubated at room temperature for 7 min, and added to the cells, to reach a final cell concentration of 1 × 106 cells/ml. All previous steps were performed in serum-free NK-MACS® medium supplemented with 1% NK-MACS® Supplements, with or without 1% Pen/Strep and 80 ng/ml IL-1β (Miltenyi Biotec), 500 IU/ml IL-2 (Miltenyi Biotec or Novartis). Finally, 10 ng/ml IL-15 (Miltenyi Biotec or Peprotech) or 50 ng/ml IL-15 (CellGenix) was added. Subsequently, the plate was centrifuged at 400 × g for 2 h at 32 °C. Twenty-four hours post-transduction half of the medium was replaced by fresh medium containing 5% human plasma and a combination of IL-2 and IL-15 [24 (link)].
7
Generating CAR-Expressing NK Cells from Cord Blood
8
Lentiviral Transduction of NK Cells
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For transduction of NK-92 cells or peripheral blood NK cells, 2.5 × 105 cells were seeded into a single well of a 24-well plate in 50 µL NK-92 or NK medium, respectively. We added 0.5 mL of the cell culture supernatant containing the lentiviral particles and 5 µL Vectofusin-1 (Miltenyi Biotec, Bergisch Gladbach, Germany) to the cells. Spinfection was performed at 400× g at 37 °C for 60 min. Afterwards, 0.5 mL of NK-92 or NK medium was added. Transgene expression was determined by flow cytometry 3 days post transduction. NK-92 cells were selected 3 days post transduction using 2 µg/mL puromycin for 2 weeks. Then, cells were used for experiments and aliquots were frozen at different time points. After thawing, cells were cultured for one week, before they were used for experiments. Peripheral blood NK cells were transduced 7 to 11 days after isolation.
9
Lentiviral Transduction of SupT1 and Raji Cells
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Next, 2 × 105 cells/well SupT1 cells were seeded in 96-well round-bottom plates, and 1 × 105 cells/well Raji cells were seeded in 48-well plates in RPMI (2 mM L-glutamine). If not otherwise noted, the cells were incubated with adapter molecules in the indicated concentrations for 30 min at 4 °C. Subsequently, LVs diluted in RPMI with 2 mM glutamine were added, and the cells were incubated at 37 °C, 5% CO2. The complete medium was supplemented 3 h (Raji) or 24 h (SupT1) after transduction. Vectofusin®-1 (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the manufacturer’s instructions. Polybrene® was diluted with the LV to a final concentration of 8 μg/mL in the transduction volume. GFP-positive cells were measured via flow cytometry 72 h (Raji) and 96 h (SupT1) post transduction [24 ].
10
Optimizing Viral Transduction with Vectofusin-1
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Vectofusin-1 (Miltenyi Biotec) was used to enhance transduction according to the manufacturer's instructions. Vectofusin-1 was reconstituted in water at 1 mg/mL and either used immediately or alternatively stored frozen in aliquots at −70°C. The reconstituted peptide was diluted in medium without serum and added to an equal volume of viral vector, also diluted in medium, and incubated for 10 min to allow viral vector-Vectofusin-1 complexes to form. The mixture was then added to the target cells such that the end concentration of the peptide was 10 or 12 μg/mL in supplemented medium. The cells were then incubated overnight at 37°C or alternatively subjected to centrifugation at 400 g for 2 h at 32°C (spinoculation) before further cultivation at 37°C. Medium was exchanged 18–24 h after transduction.
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