WM cell lines were transfected with 200 nM scrambled or 15-LOX siRNA using Amaxa® Human B Cell Nucleofector® Kit (Lonza) and Nucleofector™ 2b Device (program T-030). Cells were then incubated in complete RPMI media for 48 h. The transfection efficiency was measured by RT-PCR analysis and viable and apoptotic cells were detected by staining with Annexin/PI (Invitrogen) and subsequently flow cytometry analysis.
Amaxa human b cell nucleofector kit
Manufactured by Lonza
Sourced in Switzerland, Germany
The Amaxa Human B Cell Nucleofector Kit is a laboratory equipment product designed for the transfection of human B cells. It enables the efficient delivery of DNA, RNA, or other molecules into B cells. The kit provides all the necessary components, including solutions and cuvettes, to perform the nucleofection process.
Automatically generated - may contain errors
Lab products found in correlation
Nucleofector 2b device, by Lonza (2 mentions)
Flexitube sirna, by Qiagen (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Amaxa nucleofector 2 device, by Lonza (1 mentions)
Ex 527, by Merck Group (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Nucleofector 1 device, by Lonza (1 mentions)
Block it alexa fluor red fluorescent oligo, by Thermo Fisher Scientific (1 mentions)
Amaxa nucleofector device, by Lonza (1 mentions)
6 protocols using amaxa human b cell nucleofector kit
1
Evaluating 15-LOX Knockdown in WM Cells
2
Silencing SCF in CLL cells
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Purified CD19+ B cells from MEC1 CLL cells were transfected with 100 nM of FlexiTube siRNA directed against human SCF or 40 nM of AllStars Negative Control siRNA (Qiagen), using the Amaxa Human B Cell Nucleofector Kit (Lonza) as previously reported.26 (link) MEC1 cells were collected and processed for flow cytometry analysis at the indicated time points.
3
KRAS-Specific T Cell Activation Assay
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IFNγ ELISPOT assays were performed using BD™ ELISPOT reagents in accordance with manufacturer’s instructions66 . For assays using exogenous peptide loaded cells, target cells were incubated with 10 µM peptide (unless otherwise indicated) and co-cultured with PBMC from non-HLA*A11:01/03:01 donors in the presence of IMC-KRASG12D in RPMI-1640 medium containing 25 mM HEPES and supplemented with Penicillin-Streptomycin (Gibco™) and 10% fetal bovine serum overnight at 37 °C. For assays using iDC, capped mRNA encoding KRASWT or KRASG12D (TriLink, CA) was electroporated into iDCs using Amaxa Human B Cell Nucleofector Kit (Lonza Switzerland) with a single electrical pulse on Amaxa Nucleofector II device (Lonza) using program X-001. The dendritic cells were allowed to recover in complete AIM-V medium at 37 °C overnight and were then harvested and co-cultured with T cells. Alternatively, iDCs were pulsed with 5 µg/ml peptides (Bio-Synthesis, TX) for 1 h at 37 °C, and then co-cultured with T cells. Freshly isolated T cells were then co-cultured with iDCs at a 5:1 ratio and treated with IMC-KRASG12D in CTL test medium (CTL) overnight at 37 °C on the 96-well Strip Precoated Human IFNγ Single-Color Enzymatic ELISPOT plate (CTL). IFNγ release was quantified using the the CTL ImmunoSpot Analyzer and ImmunoSpot® software. (Immunospot Series 5 Analyzer, CTL).
4
miR-132 Modulation of B Cell Activation
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B cells (1×106) were transfected with 200 nM of miR-132 mimic, or negative control RNA (NC: cel-miR-67) which has minimum sequence identity with human miRNAs (both from Dharmacon), using the Amaxa Human B cell Nucleofector Kit (Lonza) and Nucleofector I device (Lonza), according to their standard instructions. Dose-titration experiments were carried out to define optimal concentrations of the miR-132 mimic and the negative control. Transfected cells were suspended in 500 µl of medium in 48-well plates, and cultured for 72 hours with goat anti-human IgG and IgM antibody (3.25 µg/ml) together with 6×104 of 60 Gy irradiated mouse fibroblast cell line (L cells), stably transfected with human CD40L (a gift from Dr Y. J. Liu, DNAX Research Institute of Molecular and Cellular Biology) [31] (link).
For pharmacological manipulation of SIRT1, B cells were treated with either EX-527 (a selective inhibitor of SIRT1) or with resveratrol (a small molecule activator of SIRT1), at 10 µM (both from Sigma-Aldrich) for 48 hours, together with stimulation by sCD40L (1 µg/ml) and anti-human IgG and IgM (3.25 µg/ml), as described above.
For pharmacological manipulation of SIRT1, B cells were treated with either EX-527 (a selective inhibitor of SIRT1) or with resveratrol (a small molecule activator of SIRT1), at 10 µM (both from Sigma-Aldrich) for 48 hours, together with stimulation by sCD40L (1 µg/ml) and anti-human IgG and IgM (3.25 µg/ml), as described above.
5
Silencing EZH2 in CLL B Cells
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Purified CD19+ B cells from 3 CLL cases with high EZH2 mRNA levels as determined by RQ-PCR were transfected with siRNA against human EZH2, using the Amaxa Human B Cell Nucleofector Kit (Lonza, Colonge, Germany). In detail, 5×106 CD19+ B cells from each case were resuspended in 100 μl of Human B Cell Nucleofector Solution and mixed with 70nM of 5 FlexiTube siRNAs with different sequences directed against human EZH2 (Qiagen, Hilden, Germany). In parallel, 5×106 CD19+ B cells of each patient were transfected with 40nM of AllStars Negative Control siRNA (Qiagen, Hilden, Germany). Transfections were performed using the Amaxa Nucleofector device with the U-15 program (Lonza, Colonge, Germany). Transfection efficiency was measured with the Block-iT Alexa Fluor Red Fluorescent Oligo (Invitrogen, Paisley, UK). CLL cells were collected and processed for immunoblotting and apoptosis analysis at indicated time points.
6
GPX3 Knockdown in MWCL-1 Cells
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MWCL-1 cells were transfected with 200 nM scrambled or GPX3 siRNA using Amaxa® Human B Cell Nucleofector® Kit (Lonza) and Nucleofector™ 2b Device (program T-030). Cells were then transferred to the complete RPMI media and incubated for 48 h. The transfection efficiency was measured by RT-PCR analysis and viable cells were detected by staining with a live/death cell staining solution and subsequently flow cytometry analysis.
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