Normal human dermal fibroblasts (NHDF cells; PromoCell: C12300) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a 5% CO2-humidified incubator at 37°C. 1 × 106 NHDF cells were transfected with 3.5 μg pcDNA4/TO-SHOX-WT using the Amaxa® Human Dermal Fibroblasts Nucleofector® Kit (Lonza) according to the manufacturer’s instructions. In control experiments, NHDF were transfected with 3.5 μg pcDNA4/TO-SHOX-HM, which encodes a homeodomain-mutant of SHOX leading to the amino acid exchange Y141D.
Amaxa human dermal fibroblasts nucleofector kit
Manufactured by Lonza
The Amaxa® Human Dermal Fibroblasts Nucleofector Kit is a laboratory equipment product designed for the efficient transfection of human dermal fibroblasts. It provides a standardized protocol and optimized reagents to facilitate the introduction of nucleic acids into this cell type.
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Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Nhdf cells, by PromoCell (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Human tagged orf clone, by OriGene (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
4 protocols using amaxa human dermal fibroblasts nucleofector kit
1
Overexpression of SHOX in Dermal Fibroblasts
2
NIPBL Gene Expression in Fibroblasts
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cDNAs were prepared with the Transcriptor First Strand cDNA Synthesis Kit (Roche) from total RNA from passages p4-p8 (NucleoSpin RNA Kit, Macherey-Nagel), and qRT-PCR analyses were performed using the SYBR Premix Ex Taq (Takara) and a Light Cycler 480 instrument (Roche). AnyGenes custom panels (BioNova) were used to validate some gene expression results from the microarrays. Expression was normalized with respect to the mean level of expression of the housekeeping genes ACTB1, TFAP2E, ACBD3, and LETMD1. Reactions were performed in triplicate.
To investigate the effect in gene expression upon introduction of control NIPBL (Supplementary Fig.3 ), patient 3-derived fibroblast were nucleofected (Amaxa® Human Dermal Fibroblasts Nucleofector Kit (Lonza)) with 2 µg of the commercial plasmid IDN3 (NIPBL-GFP) (NM_015384) Human Tagged ORF Clone (Origene) in triplicate, followed by qRT-PCR after 24 h.
To investigate the effect in gene expression upon introduction of control NIPBL (Supplementary Fig.
3
Culturing and Transfecting Human Cell Lines
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Human epithelial carcinoma A431 cells were grown in DMEM with 10% FBS supplemented with penicillin/streptomycin (100 U/ml each) and L‐glutamine (2 mM). Transfections of plasmids (Lipofectamine LTX with PLUS Reagent) and siRNAs (HiPerfect) were carried out according to the manufacturer's instructions. Control human primary fibroblasts were from Coriell Cell Repositories (GM01650 and GM00323, designated as controls 1 and 2 in this study), and BSCL2 patient fibroblasts S3 and S5 were established from forearm skin biopsies of patients described in Boutet et al (2009 ). Fibroblasts were cultured in MEM, with 15% non‐heat‐inactivated FBS supplemented with penicillin/streptomycin and L‐glutamine. Primary human fibroblasts were transfected with Amaxa Human Dermal Fibroblasts Nucleofector Kit (Lonza) according to the manufacturer's instructions.
4
EGFP-RAD21 Chromatin Binding Dynamics in Human Fibroblasts
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Human fibroblasts were nucleofected 18 h before acquiring microscope images with the plasmid EGFP-RAD2171 (link) using the Amaxa® Human Dermal Fibroblasts Nucleofector Kit (Lonza), following the manufacturer’s instructions. Cells were grown on glass plates. One hour before imaging, the medium was changed to one without phenol red, and 100 µg/ml of cycloheximide was added to reduce synthesis of new GFP-tagged cohesin for experiments lasting longer than 1 h. All iFRAP experiments used ten interactions of photobleaching at 100% transmission of 488 nm laser of the Carl Zeiss LSM880 confocal microscope, with a 63× NA 1.4 objective with ZEN software (black edition v2.3). Cytoplasmic and nuclear regions were bleached, leaving only half of the nuclear region unbleached. The first post-bleach frame was acquired 60 s after photobleaching to allow for complete equilibration of unbleached soluble GFP-cohesin across the nucleus. Fluorescence recovery after bleaching was quantified using ImageJ software (Fiji v1.53), considering the difference in mean intensity between bleached and unbleached regions over 1 h. The drop in mean fluorescence intensity in the unbleached nuclear region after photobleaching was used to calculate the chromatin-bound fraction of EGFP-RAD2171 (link).
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