Log In Sign up
The largest database of trusted experimental protocols

Amaxa basic nucleofector kit

Manufactured by Lonza
Visit Supplier
Sourced in Germany, United States, Switzerland

The Amaxa Basic Nucleofector Kit is a laboratory equipment product designed for the transfection of primary cells and difficult-to-transfect cell lines. It utilizes a proprietary electroporation-based technology to efficiently introduce nucleic acids into the cells. The kit includes the necessary solutions, pipettes, and other accessories required for the transfection process.

Automatically generated - may contain errors

Lab products found in correlation

Amaxa nucleofactor apparatus, by Lonza (4 mentions) Amaxa nucleofector 1, by Lonza (2 mentions) α sma, by Abcam (2 mentions) Basic fibroblast growth factor (bfgf), by R&D Systems (2 mentions) Claudin 5, by Merck Group (2 mentions) β actin, by Cell Signaling Technology (2 mentions) P gsk3β s9, by Cell Signaling Technology (2 mentions) Vegf a 164, by R&D Systems (2 mentions) Gsk3β, by Cell Signaling Technology (2 mentions) Tgf β1, by R&D Systems (2 mentions) Nuclefector 2b device, by Lonza (2 mentions) Scrambled control a and sirna sequences, by Santa Cruz Biotechnology (2 mentions) Superscript reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Quantitect pcr mastermix, by Qiagen (2 mentions) Sybr green master mix kit, by Thermo Fisher Scientific (2 mentions) Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (2 mentions) Rna stat 60, by Tel-Test (2 mentions) Pcmv6 entry vector carrying full length human hb egf plasmids, by OriGene (2 mentions) Nucleofector device, by Lonza (1 mentions) Rat ck2α, by Qiagen (1 mentions)

Spelling variants of this product

Amaxa Nucleofector (332 citations) Nucleofector kit (131 citations) Amaxa Nucleofector Kit (71 citations) Basic Nucleofector Kit (11 citations) Amaxa Basic Nucleofector Kit for Primary Mammalian smooth muscle cells (5 citations) Amaxa Basic Nucleofector Kit for Primary Mammalian Epithelial Cells (4 citations) Amaxa Basic Fibroblasts Nucleofector Kit (4 citations) Amaxa basic nucleofector kit for primary (3 citations) Amaxa kits (2 citations) Amaxa Basic Primary Neuron Nucleofector Kit (2 citations)

24 protocols using amaxa basic nucleofector kit

1

Overexpression and Knockdown of PRH and CK2 in Rat VSMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols
Expression vectors and small interfering RNAs (siRNAs) were introduced into rat aortic VSMCs utilising a Nucleofector device and the Amaxa Basic Nucleofector Kit for primary mammalian smooth muscle cells (Lonza; VPI-1004) in accordance with manufacturer's instructions. For overexpression studies, 1 × 106 cells were subjected to nucleofection with 5 μg of eGFP-encoding plasmids (control), or 2.5 μg of eGFP- and 2.5 μg of wild-type/S163C:S177C PRH-expressing plasmids using the Nucleofector D-033 programme. Similarly, gene silencing was achieved through delivery of 120–480 pmol Allstars negative control siRNAs (Qiagen; 1027281), rat/human PRH (Qiagen; SI00042056), rat CK2α (Qiagen; SI02007180, SI02007159) or rat CK2α′ (Qiagen; SI04730628, SI04730621) siRNAs. Significant depletion of PRH protein required a double knockdown, where VSMCs were Amaxa nucleofected with 240 ng Allstars negative control siRNAs or 240 ng PRH-specific siRNAs, incubated for 72 h in 10% FCS culture medium, before being subjected to nucleofection again with identical conditions. Gene transfer and silencing were validated by both RT-qPCR and Western blotting.
Wadey K.S., Brown B.A., Sala-Newby G.B., Jayaraman P.S., Gaston K, & George S.J. (2017). Protein kinase CK2 inhibition suppresses neointima formation via a proline-rich homeodomain-dependent mechanism. Vascular Pharmacology, 99, 34-44.
+ Open protocol
+ Expand
2

Measuring NHEJ and HR Repair Efficiency

Check if the same lab product or an alternative is used in the 5 most similar protocols
hNCC were stably transfected with either NHEJ reporter cassette or HR reporter cassette by nucleofection with Amaxa Nucleofector-I (Lonza program #W-01) using Amaxa Basic Nucleofector Kit for Primary Mammalian Epithelial Cells. These cassettes contain the GFP gene with recognition sequences for I-SceI endonucleases for induction of DSBs. Stably transfected cells were selected for 10 days with 1 mg/mL of G418, co-transfected (by nucleofection) with 5 μg plasmid encoding I-SceI endonuclease to induce DSBs, and GH pcDNA 3.1 or pcDNA 3.1 vector and 0.5 μg plasmid encoding DsRed (pDsRed2-N1) to control for transfection efficiency. Intact reporters were negative for GFP. Upon induction of a DSB by I-SceI digestion, the functional GFP gene was reconstituted. At 48–72h after nucleofection, the number of GFP-positive cells (corresponding to efficiency of DNA DSB repair) and DsRed-positive cells (indicating efficiency of transfection) was determined by FACS, and the ratio between GFP-positive and DsRed-positive cells used as a measure of DSB repair efficiency. NHEJ repair efficiency was 0.77–0.83 in hNCC and HR efficiency was approximately 0.05 in control cells, consistent with other reports. For each nucleofection, a minimum of 50,000 cells was analyzed by FACS and final data analysis performed using FlowJo software. Experiments were repeated at least 3 times.
Chesnokova V., Zonis S., Apostolou A., Estrada H.Q., Knott S., Wawrowsky K., Michelsen K., Ben-Shlomo A., Barrett R., Gorbunova V., Karalis K, & Melmed S. (2021). Local non-pituitary growth hormone is induced with aging and facilitates epithelial damage. Cell reports, 37(11), 110068.
+ Open protocol
+ Expand
3

Induced Pluripotent Stem Cell Generation

Check if the same lab product or an alternative is used in the 5 most similar protocols
On the first day (day 0), MEFs were transfected with various piggyBac vector combinations, using the Amaxa Basic Nucleofector Kit for primary mammalian fibroblasts (Lonza), in accordance with the manufacturer’s instructions. The piggyBac vectors (pEF1α-OddKS, pEF1α-OKddS, pEF1α-OKSdd, pEF1α-OddKddSdd, named OddKS, OKddS, OKSdd, and dd-3, respectively) were combined with CAG-PBase plasmid in a 3:1 ratio, 1.5 μg piggyBac vectors, 0.5 μg PBase, per 106 cells. Posttransfected cells were seeded onto feeder layers. After 24 hr, MEF medium was replaced by ES medium with 350 μg/ml G418 for 5 days. Different concentrations of TMP were added to test groups from the first day. The medium was refreshed every day. On day 6, medium was changed to ESC medium without G418. On day 14, colonies were either counted by GFP-positive numbers and stained by alkaline phosphatase or picked and further expanded.
Sui D., Sun Z., Xu C., Wu Y., Capecchi M.R., Wu S, & Li N. (2014). Fine-Tuning of iPSC Derivation by an Inducible Reprogramming System at the Protein Level. Stem Cell Reports, 2(5), 721-733.
+ Open protocol
+ Expand
4

Snail1 Regulation of Endothelial Cell Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cultured WT and Snail1 KO ECs were stimulated with recombinant VEGF-A/164 (100 ng/ml), bFGF (20 ng/ml) or TGF-β1 (10 ng/ml) (all from R&D Systems), and the cell lysates were subjected to immunoblot assay using antibodies against Snail1 (1:1000), Snail2 (1:1000), N1ICD (1:1000), p-ERK1/2 (Y202/204) (1:1000), p-Akt (S473) (1:500), p-GSK3β (S9) (1:2000), GSK3β (1:5000), β-actin (1:10,000) (all from Cell Signaling), Dll4 (1:1000), α-SMA (1:5000) (both from Abcam), and Claudin 5 (1:5000, Millipore). For ChIP analysis, ECs were electroporated with FLAG-Snail1 expressing vector using Amaxa Basic Nucleofector kit (Lonza) and subjected to ChIP assay as described previously 38 (link).
Wu Z.Q., Rowe R.G., Lim K.C., Lin Y., Willis A., Tang Y., Li X.Y., Nor J.E., Maillard I, & Weiss S.J. (2014). A Snail1/Notch1 Signaling Axis Controls Embryonic Vascular Development. Nature communications, 5, 3998.
+ Open protocol
+ Expand
5

Culturing Primary Tumor Cells for CRISPR Knockout

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Primary tumor cells and their derivatives were cultured in DMEM/F12 supplemented with 10% FBS, 10 ng/mL EGF, 20 mg/mL insulin, and 50 units/mL penicillin–streptomycin. The generation of FIP200f/f;PyMT;CreER cells and FIP200f/KI;PyMT;CreER cells have been described previously (27 (link),33 (link)) and deletion of Fip200 was induced by culturing with 100 nmol/L 4-hydroxytamoxifen (4-OHT). Transfection experiments were carried out using Lipofectamine 3000 Reagent (Invitrogen) for cell lines and Amaxa Basic Nucleofector Kit for electroporation for primary cells (Lonza). Production of lentivirus and transduction of cells were carried out as described previously (34 (link)). Each sgRNA sequence is as follows: sgRb1cc1 is 5’-caccg CTCCATTGACCACCAGAACC-3’. sgTbk1 is 5’-caccgCATAAGCTTCCTTCGCCCAG-3’. sgAzi2 is 5’-caccgATCTTCTACTAGCGTGTCCA -3’. MDA-MB-231 cells were obtained from ATCC. Cell lines were maintained for less than 20 passages after collection or thawing. Mycoplasma testing was performed on a monthly basis.
Okamoto T., Yeo S.K., Hao M., Copley M.R., Haas M.A., Chen S, & Guan J.L. (2020). FIP200 suppresses immune checkpoint therapy responses in breast cancers by limiting AZI2/TBK1/IRF signaling independent of its canonical autophagy function. Cancer research, 80(17), 3580-3592.
+ Open protocol
+ Expand
6

Producing anti-PEDV antibody clones

Check if the same lab product or an alternative is used in the 5 most similar protocols
According to the instructions provided by the Amaxa Basic Nucleofector Kit (Lonza, VPI-1002), HEK293 cells were transfected by electroporation. Three micrograms of pCI-anti-PEDV-VL and pCI-anti-PEDV-VH were added to 200 μL electroporation medium and mixed with approximately 1 × 104 cells and transferred into a Lonza cuvette for electroporation. After electroporation 1 mL of DMEM was added to each reaction mix then aliquoted into multiple 10 cm plates with 900 μg/mL G418 and cultured for 7 days. The G418 resistant clones with good morphology were expanded for genome extraction and cryopreservation.
Shi W., Hao H., Li M., Niu J., Hu Y., Zhao X, & Li Q. (2021). Expression and Purification of a PEDV-Neutralizing Antibody and Its Functional Verification. Viruses, 13(3), 472.
+ Open protocol
+ Expand
7

CRISPR-mediated ApoE Knockout in HEI-OC1 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Single RNAs (sgRNAs) were designed for the third exon of the mouse ApoE gene and were ligated into the PX330 plasmid containing the Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats, associated protein 9) backbone. The recombinant plasmids were transfected into HEI-OC1 cells using the Amaxa™ Basic Nucleofector™ kit (Lonza, Cologne, Germany) and viable monoclonal cell colonies were obtained by screening for G418 (geneticin, aminoglycoside antibiotic) (#A1720, Sigma, Taufkirchen, Germany). Genotyping was performed by polymerase chain reaction (PCR) sequencing (Supplementary Materials Table S1). ApoE knockout efficiency was verified with Western blotting (WB).
Ma L., Wang H., Yao J., Wei Q, & Cao X. (2022). Metabolic Abnormalities Linked to Auditory Pathways in ApoE-Knockout HEI-OC1 Cells: A Transcription-Metabolism Co-Analysis. Biomolecules, 12(9), 1217.
+ Open protocol
+ Expand
8

Silencing SOD3 in Cortical Neurons

Check if the same lab product or an alternative is used in the 5 most similar protocols
Silencing the expression of SOD3 in cortical neurons was achieved via transfection with small interfering RNA (siRNA). This procedure was performed according to manufacturer’s protocol using a Nuclefector™ 2b Device and the Amaxa™ Basic Nucleofector™ Kit for primary mammalian neurons (Lonza, Allendale, NJ). Scrambled (Control-A) and siRNA sequences directed against the rat SOD3 gene were purchased from Santa Cruz Biotechnology. Freshly isolated cortical neurons were transfected with either scrambled or SOD3 siRNA using protocol G-013. Cells were removed from cuvettes and seeded into 12-well plates containing pre-equilibrated DMEM complete medium. Media was changed to Neurobasal Complete 24 h after seeding. Suppression of SOD3 expression was confirmed using quantitative reverse-transcriptase PCR (qRT-PCR).
Davis S.M., Collier L.A., Leonardo C.C., Seifert H.A., Ajmo CT J.r, & Pennypacker K.R. (2016). Leukemia Inhibitory Factor Protects Neurons from Ischemic Damage via Upregulation of Superoxide Dismutase 3. Molecular Neurobiology, 54(1), 608-622.
+ Open protocol
+ Expand
9

Knockdown of ErbB and Nardilysin in Neural Stem Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Neurospheres were mechanically dissociated into single cells and transfected with 200 nM of siRNA designed to knock down mouse ErbB-1, ErbB-2, ErbB-3, ErbB-4 or Nardilysin genes, or with scrambled siRNA (all from Ambion, Carlsbad, CA) using Amaxa Basic Nucleofector kit (Lonza, Allendale, NJ) and the Amaxa Nucleofactor™ apparatus (Lonza, Allendale, NJ). siRNA suppression efficiency and plasmid transfection efficiency were determined by real-time PCR as follows. Total RNA from NSC was extracted using RNA STAT-60 (TEL-TEST, Friendswoods, TX) according to the manufacturer’s protocol. Aliquots of 1μg of total RNA were reverse transcribed using SuperScript Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligo-dT(18 (link))-primers (Invitrogen, Carlsbad, CA). RT-PCR amplification was performed in a 25 μl reaction containing 150 ng cDNA, 12.5 μl 2X QuantiTect PCR Master Mix (Qiagen, Valencia, CA), and 1 μl of 10 nM primers (Integrated DNA Technologies, Skokie, IL). The primers used are shown in Table 1. The real-time PCR reaction was performed using the SYBR Green Master Mix kit and the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Forster City, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control.
Wei J., Zhou Y, & Besner G.E. (2015). Heparin-Binding EGF-like Growth Factor and Enteric Neural Stem Cell Transplantation in the Prevention of Experimental Necrotizing Enterocolitis. Pediatric research, 78(1), 29-37.
+ Open protocol
+ Expand
10

Transfection of Neurospheres with HB-EGF

Check if the same lab product or an alternative is used in the 5 most similar protocols
Neurospheres were mechanically dissociated into single cells and transiently transfected with 4 μg of pCMV6-Entry vector carrying full length human HB-EGF plasmids (Origene, Rockville, MD) or with scrambled DNA control plasmid, using an Amaxa Basic Nucleofector kit (Lonza, Allendale, NJ) and the Amaxa Nucleofactor apparatus (Lonza, Allendale, NJ). Transfection efficiency was determined by real-time PCR using the following human HB-EGF primers: sense: 5′-CTCTCCCTGCCAAGTCTCAG -3′ and anti-sense: 5′-CTGCATGGAGTAGCACCAGA -3′. Transfected NSC were incubated in DMEM/F12 medium supplemented with 5% fetal bovine serum for 24h prior to administration in vivo.
Wei J., Zhou Y, & Besner G.E. (2015). Heparin-Binding EGF-like Growth Factor and Enteric Neural Stem Cell Transplantation in the Prevention of Experimental Necrotizing Enterocolitis. Pediatric research, 78(1), 29-37.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!