HL60, U937, K562, 32Dcl3 and HEK293T cells obtained from ATCC were cultured as previously described [13 (link), 23 (link), 29 (link), 42 (link)]. siMNT (Smartpool: ON-TARGET plus MNT SiRNA L-009373-00-0010), scrambled siRNA and siRNA transfection reagent DhermaFECT (ThermoScientific T-2001-03) were purchased from Dhermacon RNA technologies (Lafayette, CO, USA). shMNT (TRCN00000234786), shE6AP (TRCN0000235499) and shRNA control were purchased from Sigma. Expression plasmids for HA-MNT [43 (link)], pcDNA3.1-E6AP [10 (link)], pCAG-HA-E6AP-C843A [44 (link)], pGEX4T-GST-E6AP [45 (link)] were kind gifts from G. Meroni, Nihar Jana, Ikuo Shoji and Zafar Nawaz, respectively. Transfections in adherent cells were performed using Lipofectamine-LTX and Plus Reagent (15338-100; Invitrogen) as per manufacture`s protocol as previously described [13 (link), 42 (link)]; Transfections in myeloid cells HL60 and U937 were performed using AMAXA cell Nucleofector kit V (VCA-1003; Lonza). E6AP-C843A-HA is a catalytically inactive mutant of E6AP [13 (link)]. Cells were treated with 10μM MG132 (Z-Leu-Leu-Leu-al, Sigma, St.Louis, MO) and 10μM Lactacystin (Sigma) 6h prior to cell lysis.
Amaxa cell nucleofector kit 5
Manufactured by Lonza
Sourced in Switzerland
The Amaxa Cell Nucleofector Kit V is a lab equipment product designed for the efficient transfection of nucleic acids into a variety of cell types. It facilitates the introduction of DNA, RNA, or other macromolecules into the cell nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Amaxa nucleofector 2, by Lonza (3 mentions)
Facs aria 3 fcf, by BD (2 mentions)
Lactacystin, by Merck Group (1 mentions)
Shmnt, by Merck Group (1 mentions)
She6ap, by Merck Group (1 mentions)
Z leu leu leu al, by Merck Group (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Eclipse ti2 microscope, by Nikon (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
5 protocols using amaxa cell nucleofector kit 5
1
Establishing Cell Lines and Transfection Procedures
2
CRISPR-mediated gene editing in PLB-985 cells
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2·106 PLB-985 wild type (WT) cells were nucleofected (Amaxa Cell Nucleofector Kit V and Amaxa Nucleofector II, program C-023 (Lonza, Basel, Switzerland)) with 40 μg of the pPX458-NCF1 and a 100-nucleotide long ssODN sequence at final concentration 3 μM. The sequence of the ssODN was: GCC TCT TTG GAG GCT GAA TGG GGT CCC CCG ACT CTG GCT TTC CCC CAG GT A CAT GTT CCT GGT GAA ATG GCA GGA CCT GTC GGA GAA GGT GGT CTA CCG G. Immediately after nucleofection 500 μL of medium was added to the cuvette and the cells were incubated at room temperature for 10 minutes, transferred to 10 mL medium, then cultured for 48 hours. Additionally, the culture was supplemented with 1 μM SCR7 starting 3–4 hours post nucleofection. After 48 hours GFP expressing cells were sorted with FACS Aria III FCF (Becton Dickinson AG, Allschwil, Switzerland) into single wells with 100 μL of pre-conditioned, sterile filtered medium supplemented with 1 μM SCR7.
3
CRISPR-Cas9 Genetic Modification of PLB-985 Cells
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PX458 plasmids (15–40 μg) expressing sgRNA, Cas9, and GFP proteins were delivered into 2 × 106 PLB-985 WT or PLB-985 NCF1 ΔGT cells by nucleofection, using the Amaxa Cell Nucleofector Kit V and Amaxa Nucleofector II, program C-023 (Lonza, Basel, Switzerland), along with a 100-nt ssODN (Microsynth) at a final concentration of 3 μM (Figure 1 A). The sequence of the ssODN was: 5′-GCC TCT TTG GAG GCT GAA TGG GGT CCC CCG ACT CTG GCT TTC CCC CAG GTG TAC ATG TTC CTG GTG AAA TGG CAG GAC CTG TCG GAG AAG GTG GTC TAC C-3′. The locations of the binding sites for sgRNA are presented in Figure 1 B. Nucleofected cells were supplemented with 500 μL growth medium and incubated at room temperature for 10 min, and cells were transferred to 10 mL growth medium thereafter. 1 μM SCR7 (BioVision, Milpitas, CA, USA) was added 3–4 h after nucleofection, and cells were cultured for 48 h. GFP-positive cells were sorted into a bulk culture (FACSAria III FCF; Becton Dickinson, Allschwil, Switzerland) in preconditioned, sterile filtered growth medium supplemented with 1 μM SCR7. Sorted cells were expanded for 1 month, and individual clones were generated by limiting dilution.
4
Optimizing Transfection Efficiency with UV-Treated Plasmids
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Experiments were designed and protocols were modified based on two previous studies [43 (link),62 (link)]. An amount of 25 μl of pmaxGFP plasmid from Lonza Amaxa cell Nucleofector Kit V (Lonza, #VCA-1003) at a concentration of ~0.2 μg/μl was aliquoted into a 10 cm plate and irradiated with 600 J/m2 of UV-C light with the lid open. The same batch of UV-treated plasmids was used for testing all species to avoid batch-to-batch variation. Next, 1.2 × 106 cells were co-transfected with 6 μg of treated or untreated pmaxGFP. For each reaction, 82 μl base media and 18 μl of supplement was used to make the Nucleofector® Solution. Cell pellets were resuspended carefully in 100 μl RT Nucleofector® Solution per sample with 5 μg treated or untreated pmaxGFP added, then transferred into a certified cuvette. It was necessary for the samples to cover the bottom of the cuvette without air bubbles. Transfections were carried out with program T-027 using Amaxa Nucleofector II. Cuvettes were taken out of the holder once the program was finished. An amount of 20 μl of the mixture was added to 24-well plates with 1 ml of cell media and imaged every 1.5 hours for 3 days using a Nikon ECLIPSE Ti2 microscope with a 10X lens; the rest of the cells were added to 6-well plates with 3 ml of cell media for flow cytometry analysis.
5
Generation of G412E NOX2 Mutant
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Site-directed mutagenesis was performed by Genecust (Boynes, France) in the wild-type (WT) NOX2 complementary DNA (cDNA) subcloned into the mammalian expression vector pEF-PGKneo [11] (link) to generate the G412E mutation (c.1235G > A). The WT NOX2 and G412E NOX2 pEF-PGKneo constructs were "Endofree" purified using the Endofree plasmid Maxi Kit (QIAprep Spin Miniprep Kit, Qiagen, Courtaboeuf, France). 2 ו10 6 CYBB KO PLB-985 cells (sgRNA2, exon 3/intron 3 target) were nucleofected in presence of 2 μg of each pEF-PGKneo constructs (WT NOX2 and G412E NOX2) (Amaxa Cell Nucleofector Kit V and Amaxa
Nucleofector II, program C-023, Lonza, Basel, Switzerland). Clones expressing WT and G412NOX2 were selected by limiting dilution in medium with geneticin (1 mg/mL) for 2-3 weeks [17] (link).
Nucleofector II, program C-023, Lonza, Basel, Switzerland). Clones expressing WT and G412NOX2 were selected by limiting dilution in medium with geneticin (1 mg/mL) for 2-3 weeks [17] (link).
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