For RBM5-HA-miniAID-P2A-GFP knock-in delivery, 100 ng of the donor plasmid, and 100 ng of sgRNA/Cas9 ribonucleoprotein (RNP) were used for 500,000 SEM and MOLM13 cells. SEM cells were electroporated using the Nucleofector-4D device (Lonza) with the Lonza Cell Line Nucleofector SF solution and program EH100. MOLM13 cells were electroporated using the Nucleofector-4D device (Lonza) with the Lonza Cell Line Nucleofector SF solution and program CM138. Twenty-four hours after electroporation, cells were sorted for the GFP fluorescent marker to enrich the knock-in cell population. After the sorted cells recovered in culture for up to 3 weeks, a second sort was performed to select cells for successful knock-in by sorting for cells expressing the knock-in GFP fluorescent marker. Two weeks later, a third sort was repeated to identify single-cell-derived clones based on the high GFP expression.
Sf cell line nucleofector solution
Manufactured by Lonza
The SF Cell Line Nucleofector Solution is a transfection reagent designed for efficient delivery of nucleic acids into a variety of cell lines. It is a core laboratory equipment product that facilitates the introduction of plasmids, siRNA, or other genetic material into cells, enabling research and development applications.
Automatically generated - may contain errors
Lab products found in correlation
4d nucleofector device, by Lonza (1 mentions)
4d nucleofector, by Lonza (1 mentions)
Nebnext ultra 2 fs dna library prep kit, by New England Biolabs (1 mentions)
Renilla glo luciferase assay system, by Promega (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Amaxa 4d nucleofector, by Lonza (1 mentions)
Cts immune cell serum replacement, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
4d nucleofector x unit, by Lonza (1 mentions)
4 protocols using sf cell line nucleofector solution
1
RBM5-HA-miniAID-P2A-GFP Knock-in Protocol
2
Genome-wide CRISPR Off-target Analysis
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Genome-wide, unbiased off-target analysis was performed following the iGUIDE pipeline based on Guide-seq invented previously (62 (link),63 (link)). HEK293T cells were transfected in 20 uL Lonza SF Cell Line Nucleofector Solution on a Lonza Nucleofector 4D with program DS-150 according to the manufacturer's instructions. 300 ng of gRNA–Cas9 plasmids (or 150 ng of each gRNA–Cas9n plasmid for the double nickase), 150 ng of the effector plasmids, and 5 pmol of double stranded oligonucleotides (dsODN) were transfected. Cells were harvested after 72 h for genomic DNA using Agencourt DNAdvance reagent kit. 400 ng of purified gDNA which was then fragmented to an average of 500 bp and ligated with adaptors using NEBNext Ultra II FS DNA Library Prep kit following manufacturer's instructions. Two rounds of nested anchored PCR from the oligo tag to the ligated adaptor sequence were performed to amplify targeted DNA, and the amplified library was purified, size-selected and sequenced using Illumina Miseq V2 PE300 or V3 PE600. Sequencing data was analyzed using the published iGUIDE pipeline, with the addition of a downsampling step which ensures an unbiased comparison across samples.
3
DENV2 Replicon Luciferase Assay
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One million K562 cells were electroporated with 3 μg of DENV2-luciferase replicon [38 (link)] (provided by Jan Carette, Stanford University) in 100 μL SF Cell Line Nucleofector solution (Lonza V4XC-2012) using pulse code FF-120 (Amaxa 4D Nucleofector, Lonza). Cells were incubated for 10 minutes at room temperature following nucleofection, resuspended in 500 μL of RPMI with 7% FBS, then distributed into a 48-well plate (250 μL per well) and incubated at 37°C. At each time point, cells were lysed using the Renilla-Glo® Luciferase Assay System (Cat #E2710, Promega) according to manufacturer suggestions, and frozen at −20°C. Samples from each timepoint were then concurrently processed using the Renilla-Glo® Luciferase Assay System according to manufacturer instructions, and analyzed on a plate reader (Infinite M1000 Pro, Tecan).
4
MAIT Cell TCR Electroporation Protocol
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The mRNA for TCR H4 specific for the HCV NS3 1073-1081 epitope was transcribed in vitro using the mMessage mMachine T7 Ultra kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. On day 19–21 of the MAIT cell expansion culture, 1 × 107 MAIT cells were suspended in 100 μL supplemented SF Cell Line Nucleofector Solution (Lonza) with 20 μg mRNA encoding the TCR. The suspension was transferred to a Nucleocuvette vessel and electroporated in a 4D-Nucleofector X Unit (Lonza). Immediately after pulsing, 400 μL prewarmed ImmunoCult Human T cell Expansion Medium (STEMCELL) supplemented with 8% CTS Immune Cell Serum Replacement (Thermo Fisher Scientific), 50 ng/mL animal-free recombinant human IL-2 (Peprotech), 100 μg/mL Normocin (MilliporeSigma), and 100 U/mL Penicillin/Streptomycin (GE Healthcare) were added to the cuvette and incubated at 37°C for 5 minutes. The contents of the cuvette were further collected to a final volume of 3 mL using the same medium and incubated overnight. 18–24 hours after electroporation, MAIT cell TCR expression was quantified by flow cytometry using a FITC-labeled anti-mouse Vβ8.3 antibody (BD Biosciences).
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