NK cells were isolated from PBMCs and expanded using a GMP-compliant protocol with irradiated EBV-LCL feeder cells as previously described [23 (link)]. Retroviral particles containing the pMSGV1-CXCR2 vector were recovered from supernatant of confluent cultures of PG13 packaging cells, kindly provided by Dr. Patrick Hwu (University of Texas M.D. Anderson Cancer Center, USA). Retrovirus containing the vector pMSGV1-NGFR-N which encodes the human nerve growth factor receptor gene was used as control. NK cells expanded for eight to 10 days and confirmed to be pure from feeder cells by flow cytometry were transduced using RetroNectin reagent (Takara Bio) following the manufacturer’s protocol. Briefly, viral supernatant was bound to RetroNectin-coated 6-well plates by 2 hour centrifugation at 32 °C at 2000 x g. After virus removal, NK cells were added to the wells at 0.5 × 106/mL in X-Vivo 20 medium containing 10% human AB serum and 1000 IU/mL IL-2 and centrifuged at 1000 x g for 10 min. Viral spinoculation was repeated on the following day to improve transduction efficiency. The next day, NK cells were pooled and cultured at a concentration of 1 × 106/mL supplemented with 500 IU/mL IL-2 for two to 3 days. For the migration experiments, transgene-positive NK cells were isolated by positive selection with anti-APC beads (Miltenyi Biotech) with >90% purities on average.
Retronectin reagent
Manufactured by Takara Bio
RetroNectin reagent is a recombinant human fibronectin fragment that enhances the efficiency of gene transduction. It is designed to facilitate the attachment and activation of target cells during the gene transfer process.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Pcl eco vector, by Addgene (2 mentions)
Lipofectamine, by Thermo Fisher Scientific (2 mentions)
Anti apc beads, by Miltenyi Biotec (1 mentions)
Lipofectamine ltx with, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Plko 1 vector, by Addgene (1 mentions)
Panserin medium, by PAN Biotech (1 mentions)
Anti phycoerythrin pe microbeads, by Miltenyi Biotec (1 mentions)
Cts optmizer medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by BioLegend (1 mentions)
Stemspan sfem serum free media, by STEMCELL (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Facsaria, by BD (1 mentions)
Pvsv g vector, by Takara Bio (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
96 well plate, by Corning (1 mentions)
Lenticrispr v2 plasmid, by Addgene (1 mentions)
14 protocols using retronectin reagent
1
Retroviral Transduction of Expanded NK Cells
2
Lentiviral Vector Production and Transduction
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The mouse pGIPZ‐shRNA library lentiviruses and pLKO shRNA lentiviruses were prepared as previously described 30 , 31 . Briefly, the lentiviral vectors were transfected into 293FT cells together with pPAXs and pMD2 vectors using Lipofectamine® LTX with Plus™ Reagent (ThermoFisher Scientific, #15338100). The viral supernatant was collected 48 h post‐infection and concentrated by ultracentrifugation. The concentrated viruses were divided into aliquots and snap frozen in liquid nitrogen before freezing at −80°C. Transduction of iHoxB4 cells was carried out as previously described 30 . Transduction of human CD34+ cells were performed using RetroNectin reagent (TaKaRa).
3
Generating SWELL1 Knockdown Cell Lines
4
Lentiviral Transduction and Cell Sorting
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HL60 cells were transfected using empty (lv-vector) or ECRG4 (lv-ECRG4) lentivirus using RetroNectin reagent (Takara Inc.) as described by the manufacturer. Five days later, GFP+ cells were sorted at the cell sorting facility of the Moores Cancer Center at UCSD and expanded. Two weeks later, GFP+ cells were selected a second time by cell sorting, expanded, and passaged, and aliquots of both sorts were frozen in serum–dimethyl sulfoxide.
5
Transduction of Cryopreserved PBMCs
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Cryopreserved PBMCs from four unrelated FHL3 patients (Table S1 ) and healthy donors were cultured in Panserin medium (PAN Biotech; Aidenbach, Germany) supplemented with 5% human AB serum (Dutscher), 100 IU/ml human pro-interleukin-2 (pro-IL2; Novartis) and 1% penicillin- streptomycin at a density of 1.5 × 106 cells/ml. Cells were stimulated with anti CD3-CD28 coated-magnetic beads (Dynabeads Human T-activator) for 48 h in 48-well plates coated with 250 μl (25 μg/ml) RetroNectin reagent (CH296, Takara). Concentrated vector was added at 100 multiplicity of infection (MOI) and incubated overnight at 37°C with the cells at a density of 2 × 106 cells/ml in presence of 0.1 μg/ml LentiBoost (SIRION Biotech). The cells were then washed and seeded into culture plates in the presence of 100 IU/ml pro-IL2 for 5 days prior to functional analyses.
6
Lentiviral CAR-T Cell Generation
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Fresh or thawed T cells were activated for 24 hours on an OKT3 (1 μg/ml) and CD28 (1 μg/ml) antibody-coated 24-well plate in CTS OpTmizer medium (Gibco) supplemented with OpTmizer T cell expansion supplement, 10% FBS, 1% l -glutamine (200 mM), 1% penicillin (100 U/ml), 1% streptomycin (100 μg/ml), IL-7 (10 ng/ml), and IL-15 (5 ng/ml). Activated T cells were then transduced on non–tissue culture–treated plates coated with RetroNectin reagent (Takara Bio) (19 μg/ml) and CAR lentivirus (multiplicity of infection of approximately 10) according to the manufacturer’s instructions. CAR-T cells were then expanded for at least 7 days before use in experiments. Before all experiments, CAR-T cell transduction efficiency was normalized to 50% CAR+ cells (for in vitro cytotoxicity screening assay) or to the lowest efficiency donor by adding of non-transduced T cells. If necessary, CAR-T cells were purified using anti-phycoerythrin (PE) microbeads (Miltenyi Biotec) following anti-EGFR-PE staining according to the manufacturer’s instructions.
7
Conditional C/EBPα Knockout in Mouse Hematopoiesis
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C/EBPα f/f Mx1-Cre + mice were intraperitoneal injected 30 µg Poly I:C (Sigma-Aldrich, Cat. No. P0913) per gram of mouse weight every alternative day for 18 days to induce C/EBPα conditional knockout. Two tibia, two femur and two pelvis were harvested from each knockout mice. Bone marrow cells were isolated from each bones and proceed to lineage depletion using direct lineage depletion kit (Milenyi Biotec, . Cells were then stained with antibody (Supplementary Table . 2) and LSK cells were isolated through cell sorting using FACSAria (BD). Sorted LSK cells were cultured in StemSpan SFEM serum free media (stemcell technologies) contains 40 ng/ml mSCF (Biolegend, Cat. No. 579702), 40 ng/ml mTPO (Biolegend, Cat. No. 593302) and 10 ng/ml IL3 (Biolegend, Cat. No. 575502) for virus transduction. 200 MOI retrovirus and 5 µg/cm 2 RetroNectin reagent (Takara, Cat. No. T100A) were used in the transduction following the manufactory protocol. 48 hours post transduction, 1 × 10 5 LSK cells per mouse were injected into lethally irradiated (1000 cGy) B6.SJLPtprca Pepcb/BoyJ (Pep Boy) congenic recipients along with 1 × 10 5 lineage depleted Pep boy bone marrow cells for radioprotection.
8
TCRa/b Transduction of Jurkat and CD8+ T Cells
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Each TCRa-and b-transduced pMSCV vector created by VectorBuilder (Chicago, IL) and pVSV-G vector (TaKaRa) were transfected into packaging cells using Lipofectamine 3000 Reagent (Thermo Fisher Scientific). After 48 hours, the supernatant was concentrated and transfected into the NFAT-Jurkat cell line or CD8 + T cells from healthy donors using RetroNectin Reagent (TaKaRa).
9
Lentiviral Transduction of CD34+ HSCs
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Lentiviral particles were produced in HEK293T cells. In each 150 mm tissue culture dish, 15 μg of the lentiviral vector was transiently packaged with 9 μg psPAX2 and 6 μg pMD2.G with JetPrime (Polyplus). Viral supernatant was collected at 48 h post-transfection. Virus concentration was achieved by ultracentrifugation at 30,000 rpm through a 20% sucrose cushion for 120 min at 4 °C. For infection of CD34+ cells, each well of non-tissue-culture-treated 96-well plates (Corning) was coated with 6.4 μg RetroNectin reagent (Takara Clontech) in 100 μl PBS and incubated overnight at 4 °C. The coated wells were washed and blocked with PBS, containing 2% BSA, for 30 min at RT. Viral particles (multiplicity of infection 50 for OE and 100 for KD) were added to the RetroNectin-coated wells and centrifuged at 2,000g for 2 h at 32 °C. The supernatant was removed, and wells were washed once with PBS containing 2% BSA. Then, 0.5–1×104 CD34+ cells (24 h pre-stimulated in HSC medium with UM171) were placed into lentivirus-preconditioned wells. At 16 h post-transduction, cells were washed from the RetroNectin with HSC medium and placed onto a fresh plate.
10
Retroviral and Lentiviral Vector Production
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