HEK293T-GFPΔ35, U2OS-GFPΔ35 and C6 were seeded onto 96-well plates at a density of 2 × 105 cells per well in serum-containing medium with 200 ng/ml of nocodazole (Sigma-Aldrich). After 30 min, the AAV donor vector (t37EGFP-AAV1 for HEK293T-GFPΔ35 and U2OS-GFPΔ35, and nestin-AAV1 for C6) was added onto cells in the presence of 200 ng/ml of nocodazole. After 16 h, cells were dissociated with 0.05% trypsin (Thermo-Fisher), centrifuged at 400 × g for 3 min, washed once with PBS and resuspended in 20 μl of Nucleofector Solution SF (Lonza) with 10 μl of RNP. Cells were nucleofected using the Amaxa 96-well Shuttle system (Lonza) and the program CM120. Immediately after nucleofection, 100 μl of serum-containing medium was added to nucleofected wells, and cells were transferred into a fresh 96-well plate until further use. HEK293T-GFPΔ35 and U2OS-GFPΔ35 were maintained in serum-containing medium, and C6 cells were maintained in serum-containing medium with 50 ng/ml of FGF-2.
Amaxa 96 well shuttle system
Manufactured by Lonza
The Amaxa 96-well Shuttle System is a laboratory equipment designed for the efficient transfection of cells in a 96-well format. It provides a automated and high-throughput method for introducing nucleic acids, such as plasmids or siRNA, into a variety of cell types.
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Lab products found in correlation
Rat tail collagen type 1, by BD (2 mentions)
Cell line buffer sf, by Lonza (2 mentions)
4d x unit, by Lonza (2 mentions)
Snls spcas9 snls, by Aldevron (2 mentions)
Sg cell line nucleofector solution, by Lonza (2 mentions)
Sgrna, by Synthego (2 mentions)
Nocodazole, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Nucleofector solution sf, by Lonza (1 mentions)
Jetprime transfection reagent, by Polyplus Transfection (1 mentions)
K562 cells, by Lonza (1 mentions)
K562 cell, by American Type Culture Collection (1 mentions)
Am4611, by Thermo Fisher Scientific (1 mentions)
Sf solution, by Lonza (1 mentions)
Pre mir s, by Thermo Fisher Scientific (1 mentions)
Facscalibur system, by BD (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Six well dishes, by BD (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
On targetplus smartpool sirna, by Horizon Discovery (1 mentions)
11 protocols using amaxa 96 well shuttle system
1
Optimized CRISPR-AAV Transduction Protocols
2
Isolation and Manipulation of Mouse Keratinocytes and Sebocytes
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Primary mouse keratinocytes were isolated from dorsal skin as previously described (Jensen et al, 2010 ) and cultured on confluent irradiated 3T3‐J2 fibroblast feeders in calcium‐free FAD medium (DMEM: Ham's F12, 3:1, 1.8 × 10−4 M adenine) supplemented with 10% fetal calf serum, hydrocortisone (0.5 μg ml−1), insulin (5 μg ml−1), cholera toxin (8.4 ng ml−1), and epidermal growth factor (10 ng ml−1). siRNAs for the negative control and mouse Lef1 (Ambion) were introduced into cells by nucleofection using the Amaxa 96‐well shuttle system (Lonza) as previously described (Mulder et al, 2012 ).
The SebE6E7 sebocyte line was obtained and cultured as described previously (Lo Celso et al,2008 ). All cell stocks were routinely tested for mycoplasma contamination and were negative. SebE6E7 cells were transfected with a control plasmid or human full‐length or ΔN34Lef1 plasmids (Takeda et al, 2006 ) using jetPRIME transfection reagent (Polyplus transfection) following the manufacturer's instructions.
The SebE6E7 sebocyte line was obtained and cultured as described previously (Lo Celso et al,
3
Generation of Engineered K562 Cell Lines
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K562 cells (ATCC, Manassas, VA; catalog number CCL-243) were engineered to have an NC CCR5 target site with either a 6 or 7 bp spacing or a canonical CCR5 site inserted into the gene bearing the AAVS1 safe harbor43 (link). Analogous cells lines were also generated that inserted an NC AAVS1 target site with either a 6 or 7 bp spacing or a canonical AAVS1 site into the CCR5 target site. Transfections were performed by combining 200 ng of DNA for each expression plasmid with 2E5 K562 cells and 2 µM of a single-stranded oligo donor using the Amaxa 96-well Shuttle System (Lonza, Allendale, NJ) as per the manufacturer’s protocol (oligo sequences in Supplementary Table 1 ). DNA-PK inhibitor NU7441 (Cayman Chemical, Ann Arbor, MI) was added at a concentration of 2 µM at 4 and 20 h post transfection. Three days post transfection, single-cell clones were generated by pelleting and resuspending cells at a concentration of 0.3 cells/200 µl media and 200 µl was put into each well of two 96-well plates. After 2 weeks of incubation, a sample was taken from each single-cell clone for sequencing the modified loci via PCR followed by deep sequencing using an Illumina MiSeq (Illumina, San Diego, CA). The cell lines with the correct DNA sequence inserted were consolidated, frozen with 5% dimethyl sulfoxide, and stored in liquid nitrogen.
4
Knockdown of DLL1 in Human Keratinocytes
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siRNA nucleofection was performed with the Amaxa 96-well shuttle system (Lonza)35 (link). Pre-confluent human keratinocyte cultures were disaggregated and re-suspended in cell line buffer SF (Lonza). For each 20 μl transfection (program FF-113) reaction, 2 × 105 cells were mixed with 1 μM siRNA duplexes as described previously33 (link),35 (link). Transfected cells were allowed to recover at ambient temperature for 10 min and were subsequently re-plated onto rat-tail type I collagen (20 µg ml−1 in PBS, BD Biosciences)-coated cell culture well plates (Falcon) and grown in KSFM for 24 h before being used for downstream assays35 (link). 27mer siRNA oligo duplexes (SR509346, Origene) were used for gene knockdown of human DLL1 (the sequences of siRNA oligos can be found in Supplementary Table 4 ). Non-targeting control siRNAs were from Ambion (AM4611 and AM4637).
5
Transfecting K562 cells with ZFNs
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Transfections into K562 cells (either wild-type or engineered cells) were performed by combining 2E5 cells with either plasmid- or mRNA-encoding ZFNs and SF solution (Lonza) using the Amaxa 96-well Shuttle System as per the manufacturer’s protocols. Following nucleofection, cells were placed into RPMI media containing 10% fetal bovine serum. A variation of a transient cold shock protocol74 (link) was employed by placing the 96-well plate of cells at 30 °C for 24 h followed by incubation at 37 °C for an additional 48 h. Cells were then pelleted and genomic DNA was isolated using QuickExtract (Lucigen, Middleton, WI) as per the manufacturer’s instructions. Target loci were then analyzed by PCR amplification followed by deep sequencing (MiSeq).
6
RNP Complex Formation and Nucleofection
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To prepare ribonucleoprotein (RNP) complexes, reconstituted sgRNA (Synthego) and then sNLS-SpCas9-sNLS (Aldevron) were added to complete SG Cell Line Nucleofector Solution (Lonza), to a final volume of 20 μl. The mixture was incubated at room temperature for 15 min to allow RNP complexes to form. A Cas9:sgRNA molar ratio of 1:2 was used, unless otherwise noted. Total RNP doses described refer to the amount of the limiting complex member (Cas9). To nucleofect, 1.5 × 105 cells were harvested, washed with PBS, resuspended in 20 μl of RNPs and electroporated using the Amaxa 96-well Shuttle System or 4D X Unit (Lonza) and program EN-138.
7
Nucleofection of miR-9 in L2.3 Cells
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PremiRs (Life Technologies) for miR-9 were nucleofected into L2.3 cells using Rat Neuron 96-well Nucleofector Kit (VHPG-1003) in conjunction with an amaxa 96-well shuttle system (Lonza, Cologne, Germany). Observed transfection efficiencies using the 96-well shuttle system were consistently >95% for both the L2.2 and L2.3 NSCs. At four hours after transfection, L2.3 cells were differentiated by removing FGF2 from the growth medium and allowed to differentiate for 72 hours. At 3 days, cells were fixed with 2% paraformaldehyde and analyzed for anti-βIII-tubulin (TuJ1, 1∶500, Covance) expression by flow cytometry on the FACSCalibur System (BD Biosciences, San Jose CA). Data was analyzed using BD's CellQuest Pro v. software.
8
Efficient siRNA Nucleofection in Keratinocytes
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siRNA nucleofection was performed with the Amaxa 96-well shuttle system (Lonza). Pre-confluent NHK cultures were disaggregated and resuspended in cell line buffer SF (Lonza). For each 20 μl transfection (program FF-113) reaction, 2 × 105 cells were mixed with 1 μM siRNA duplexes as described previously15 (link). Transfected cells were allowed to recover at ambient temperature for 10 min and were subsequently re-plated into rat-tail type I collagen (20 μg ml−1 in PBS, BD Biosciences)-coated cell culture well plates (Falcon) and, unless stated otherwise, grown in KSFM medium for 24 h before being used for downstream assays. SMARTpool ON-TARGET plus siRNAs (GE Dharmacon) were used for gene knockdown. Each SMARTpool was a mix of four sets of RNAi oligos (the sequences of siRNA oligos can be found in Supplementary Table 3 ). Non-targeting control siRNAs were from Ambion (AM4611 and AM4637). SCC13 cells (5 × 105) were reverse transfected with siRNAs (25 pmol) in six-well dishes (Falcon) using Lipofectamine RNAiMAX reagent in combination with Opti-MEM medium (Life Technologies) according to the manufacturer's instructions.
9
Efficient RNP Delivery to Cell Lines
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See detailed protocol in Supplementary Materials. Briefly, to prepare RNP complexes, reconstituted sgRNA (Synthego) and then sNLS-SpCas9-sNLS (Aldevron) were added to complete SG Cell Line Nucleofector Solution (Lonza), to a final volume of 20 µL. The mixture was incubated at room temperature for 15 minutes to allow RNP complexes to form. A Cas9:sgRNA molar ratio of 1:2 was used, unless otherwise noted. Total RNP doses described refer to the amount of the limiting complex member (Cas9). To nucleofect, 1.5 x 10 5 cells were harvested, washed with PBS, resuspended in 20 µL of RNPs, and electroporated using the Amaxa 96-well Shuttle System or 4D X Unit (Lonza) and program EN-138.
10
CRISPR-Mediated Genome Editing Workflow
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Cells were detached by 0.05 % trypsin or a gentle dissociation reagent and spun down at 600 g for 3 min, then washed with PBS. Nucleofection was then conducted using an Amaxa 96-well Shuttle system following the manufacturer’s protocol, using 10 µL of Cas9 RNP and DNA donor (Cas9 : 100 pmole, gRNA : 120 pmole, DNA donor : 100 pmole) (1.6 mg/mL of Cas9 protein and later diluted to 16 µg/mL in cell culture). The nucleofection program was chosen to match the cell type used for the experiment. After the nucleofection, 500 µL of growth media was added, and the cells were incubated at 37°C in tissue culture plates. The cell culture media was changed 16 hr after the nucleofection, and the cells were incubated for a total of 3 days before genomic DNA extraction and analysis was conducted.
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