Plasmid DNA was prepared using the Qiagen Maxi prep kit according to the manufacturer’s instructions. Plasmid pcDNA3.1 was obtained from Stratagene. pDUAL CMMP(-) constitutively active NFATc1 and NFATc2 was a gift from Dr. Anjana Rao (La Jolla Institute) through Dr. Alan Attie at the University of Wisconsin. pcDNA3.1-constitutively active CAMKIV was a gift from Dr. Xiang-Jiao Yang (McGill University) [88 (link)]. Akata Burkitt lymphoma cells were nucleofected using the Amaxa Nucleofector 2b device (Lonza) and program A-030 (with Buffer V) in 12-well dishes with 1 μg of vector control, constitutively active NFATc1, constitutively active NFATc2, or constitutively active CAMKIV plasmid. The cells were washed with PBS and harvested in SUMO buffer 24 hrs post-nucleofection as described above. Cell extracts were subjected to immunoblot analysis as described above. pHEBo-MT-EBNA2 constructs were constructed as previously described [28 (link)]. P3HR1 BL cells were nucleofected using the Amaxa Nucleofector 2b device (Lonza) and program A-030 (with Buffer V) in 12-well dishes with 1 μg of T1 EBNA2 or T2 EBNA2. At 3 days post-nucleofection, cells were treated with 200μg/mL of hygromycin and continually passaged until stable cell line was produced. Stable cells were treated with 10 or 20 μM CdCl2 for 24–48 hours and harvested in SUMO buffer as described above.
Amaxa nucleofector 2b device
Manufactured by Lonza
Sourced in Switzerland
The Amaxa Nucleofector 2b device is a lab equipment product designed for the efficient transfection of cells. It utilizes a proprietary Nucleofection technology to deliver nucleic acids, such as DNA or RNA, into a wide range of cell types, including primary cells and difficult-to-transfect cell lines. The core function of the Amaxa Nucleofector 2b device is to enable researchers to introduce genetic material into cells for various applications, including gene expression studies, gene silencing, and cell line engineering.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Human stem cell nucleofector kit 1, by Lonza (3 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Basic nucleofector kit for primary mammalian epithelial cells, by Lonza (2 mentions)
Accutase, by Merck Group (2 mentions)
Monolight 3010 luminometer, by BD (2 mentions)
Reporter lysis buffer, by Promega (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Nucleofector kits for human t cells, by Lonza (1 mentions)
On targetplus nontargeting control pool, by GE Healthcare (1 mentions)
Nucleofector kit 5, by Lonza (1 mentions)
Nucleofector kit c, by Lonza (1 mentions)
Bmi1 sirna, by Thermo Fisher Scientific (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Nucleofector solution 5, by Lonza (1 mentions)
Pmaxgfp, by Lonza (1 mentions)
Cell line nucleofector kit 5, by Lonza (1 mentions)
Puromycin dihydrochloride, by Thermo Fisher Scientific (1 mentions)
Geneticin g418 sulfate, by Thermo Fisher Scientific (1 mentions)
Amaxa human stem cell nucleofector kit, by Lonza (1 mentions)
34 protocols using amaxa nucleofector 2b device
1
Plasmid DNA Preparation and Transfection
2
SiRNA Transfection of Primary T Cells
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For experiments involving use of siRNA, 5–12 × 106 CD25−CD4+ T cells from individual donors were resuspended in 100 µl of Nucleofection buffer solution for human primary T cells (Nucleofector Kits for Human T Cells, Lonza) containing 2 µM of ON‐TARGETplus PPP1R11 siRNA pools or ON‐TARGETplus nontargeting control pool (both Dharmacon, GE Healthcare). To deconvolute the effect of the siRNA pool, confirmation experiments were also performed with 2 individual PPP1R11 siRNAs (contained in the pool of 4 siRNAs above), which are denoted as PPP1R11‐07 and PPP1R11‐08. The cells were transfected using program U‐014 of the Amaxa Nucleofector™ 2b device using the manufacturer's recommendations. Following nucleofection, the cells were transferred to prewarmed X‐VIVO 15 medium and incubated for 4.5 days at 5% CO2/37°C unless otherwise mentioned. The medium was changed once following 5 h of incubation. The cells were either directly frozen for studying the unstimulated state or stimulated with particular stimulations for specific time periods depending on the nature of further analyses.
3
siRNA Knockdown via Nucleofection
4
Efficient BMI-1 Knockdown in Leukemia Cells
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MOLM-13 and U-937 cells were transfected with small interfering RNA (siRNA) oligonucleotides with the Amaxa Nucleofector 2b device, using the Nucleofector Kit C (program X-001 for MOLM-13 cells and W-001 for U-937 cells; Lonza, Basel, Switzerland). Cells were transfected with negative control siRNA or with BMI-1 siRNA (HSS101040; Life Technologies, Carlsbad, CA, USA).
5
Transient Transfection of Colon Cells
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Normal diploid colon epithelial cells (HCEC-1CT) were transiently transfected through electroporation with 5 μg of pCMV empty vector (Con) or wild-type pCMV6M-PAK1 (WT-PAK1) plasmid DNA, a kind gift from Jonathan Chernoff, Fox Chase Cancer Center, Philadelphia, PA. HCEC-1CT cells were electroporated using the Amaxa nucleofector 2b device with program number T023 and basic Nucleofector Kit for primary mammalian epithelial cells (Lonza) according to manufacturer's instructions.
6
Knock-in of mRuby4(11) into HIST2H2BE locus
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For knock-in of mRuby411 into the HIST2H2BE locus, we ordered 200-nt HDR templates in single-stranded DNA (5′-gcccggcgagctggccaagcacgccgtgtccgagggcaccaaggcggtcaccaagtacaccagctccaagGGTGGCGGCGAAACCTACGTAGTGCAAAGAGAAGTGGCAGTTGCCAAATACAGCAACtgagtccctgccgggacctggcgctcgctcgctcgagtcgccggctgcttgactccaaaggctcttttcagag-3′, Integrated DNA Technologies). Cas9 protein was expressed in E. coli and purified by the Kipreos laboratory at UGA as described previously11 (link). sgRNA and Cas9/sgRNA ribonucleoprotein complexes were prepared as described before12 (link). After the treatment of HEK293 FT cells with nocodazole (200 ng/mL, Sigma-Aldrich) for 16 h, we performed electroporation on an Amaxa Nucleofector 2b device with Nucleofector Solution V reagents (Lonza).
nocodazole-treated cells were resuspended at a concentration of 1 × 104 cells/μL in 100 μL of Nucleofector Solution V. We added cells to the RNP/donor template mixture (50 μL), electroporated using the Q-001 program, and quickly transferred to 12-well plates with pre-warmed media. Electroporated cells were cultured for 2–5 days and transfected with mRuby41-10 plasmid.
nocodazole-treated cells were resuspended at a concentration of 1 × 104 cells/μL in 100 μL of Nucleofector Solution V. We added cells to the RNP/donor template mixture (50 μL), electroporated using the Q-001 program, and quickly transferred to 12-well plates with pre-warmed media. Electroporated cells were cultured for 2–5 days and transfected with mRuby41-10 plasmid.
7
Overexpression of AP4B1 Variants in SH-SY5Y Cells
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AP4B1 plasmid DNA constructs were purchased from Twist Bioscience (Supplementary File 2 ). Plasmid to express pmaxGFP™ was purchased from Lonza (Lonza, #VCA-1003). Nucleofection of AP4B1KO SH-SY5Y cells with full-length wild type AP4B1 or AP4B1 carrying variants found in PVUS/VUS_01 and PVUS/VUS_02 was carried out using the Cell Line Nucleofector Kit V (Lonza, #VCA-1003) and Amaxa Nucleofector 2b device (Lonza, #AAB-1001). Per 2 × 106 cells, 2 μg of AP4B1 plasmid and 2 μg of GFP plasmid were used. After nucleofection cells were resuspended in differentiation medium with retinoic acid and seeded onto 96-well plates (Greiner Bio-One, Cat# 655090) at a density of 14 × 103 per well. After 48 h, cells were fixed using 4% paraformaldehyde (PFA) and stained and imaged as described below.
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AP4B1 plasmid DNA constructs were purchased from Twist Bioscience (
8
Targeted Genome Editing in Stem Cells
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All reporter lines were generated by nucleofection of the plasmid DNA with the Amaxa nucleofector 2b device (Lonza, AAB-1001) and Human Stem Cell Nucleofector Kit 1 (Lonza, VPH-5012) following the manufacturer’s guidelines. For each nucleofection 106 single cells were used. For genotyping, DNA was extracted using the QuickExtract DNA Extraction Solution (Cambio QE09050) and a PCR assay was performed to identify correctly edited clones. See Supplementary file 1 for oligo sequences used for genotyping. Genomic integrity of the final clones was confirmed by SNP arrays (Supplementary file 2 ).
Insertion into the AAVS1 safe harbor locus was performed using TALEN technology as described before (Bagley et al., 2017 (link); Hockemeyer et al., 2011 (link)). For this purpose, the nucleofection mix containing 0.5 μg of each of the TALEN plasmids and 1.5 μg of each of the donor plasmids was used. Nucleofected cells were grown for 4–7 days and then selected with appropriate antibiotics. For puromycin resistance reporter: 1 µg/ml puromycin dihydrochloride (Thermo Fisher Scientific, A1113803) and for neomycin resistance reporter 250 µg/ml geneticin/ G418 sulfate (Thermo Fisher Scientific, 10131035). Surviving colonies were picked manually, transferred into 24-well plates and further expanded for genotyping and cryopreservation.
Insertion into the AAVS1 safe harbor locus was performed using TALEN technology as described before (Bagley et al., 2017 (link); Hockemeyer et al., 2011 (link)). For this purpose, the nucleofection mix containing 0.5 μg of each of the TALEN plasmids and 1.5 μg of each of the donor plasmids was used. Nucleofected cells were grown for 4–7 days and then selected with appropriate antibiotics. For puromycin resistance reporter: 1 µg/ml puromycin dihydrochloride (Thermo Fisher Scientific, A1113803) and for neomycin resistance reporter 250 µg/ml geneticin
9
TFAM Knockout and Knockin in hiPSCs
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TFAMKI cells were generated by electroporation of AAVS1-guide (2 μg) and template vector (4 μg) with an Amaxa nucleofector 2b device (program B-016) and Amaxa human stem cell Nucleofector kit (Lonza). Cells were selected by G418 (SANTA CRUZ, sc-29065A, 200 μg/mL) for five days, and the remaining cells were split into 6-well plates for colony picking in mTeSR1 medium supplemented with Y-27632 (10 μM). Expanded clones were selected by G418 again. Genotyping PCR was performed to detect whether Exo-TFAM has been knocked in.
TFAM-/- cells were generated by electroporation of TFAM gRNA2 (4 μg) as above. Cells were selected by puromycin (0.5 μg/mL) for one day, and the survived cells were cultured with Dox (100 ng/mL, Sigma) until the cells were split into 6-well plates in mTeSR1 medium supplemented with Y-27632 (10 μM) and Dox (Sigma, D9891, 100 ng/mL). Single colonies were then picked up and genotyped by PCR, followed by Sanger sequencing.
The guide sequence information was as follows:
TFAM-g1 (5′-CACCCGTTTCTCCGAAGCATGT-3′); TFAM-g2 (5′-CACCGCAGAGCTGTGCACCGGCTG-3′); AAVS1-g (5′-CACCGGGGCCACTAGGGACAGGAT-3′).
TFAM-/- cells were generated by electroporation of TFAM gRNA2 (4 μg) as above. Cells were selected by puromycin (0.5 μg/mL) for one day, and the survived cells were cultured with Dox (100 ng/mL, Sigma) until the cells were split into 6-well plates in mTeSR1 medium supplemented with Y-27632 (10 μM) and Dox (Sigma, D9891, 100 ng/mL). Single colonies were then picked up and genotyped by PCR, followed by Sanger sequencing.
The guide sequence information was as follows:
TFAM-g1 (5′-CACCCGTTTCTCCGAAGCATGT-3′); TFAM-g2 (5′-CACCGCAGAGCTGTGCACCGGCTG-3′); AAVS1-g (5′-CACCGGGGCCACTAGGGACAGGAT-3′).
10
Generating mScarlet-VCL Fusion Protein
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1·106 cells were electroporated with 4 µg mScarlet donor plasmid DNA using Amaxa Nucleofector™ 2b Device (Lonza) and immediately seeded in 5 ml of the complete medium on one well of a 6-well plate and incubated at 37°C, 5% CO2 for 6 h. Dead and non-adherent cells were discarded by washing twice with 1 ml of complete medium and attached cells were transduced using VCL-targeting gRNA expressing lentiviral vectors, as described above. The next day, supernatant was removed, and cells were gently washed with fresh complete medium. Antibiotic selection with G418 (Thermo Fisher Scientific) was started 48 h post-transduction and continued for at least 7 days. Finally, mScarlet-positive cells were sorted using FACS. At least 300 mScarlet-positive cells were collected for the generation of each variant. The mScarlet-VCL recombination was confirmed by western blotting, fluorescence microscopy and sequencing.
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