Human fetal lung fibroblasts were transfected with 3 independent 50nM ON-TARGETPlus siRNAs (Dharmacon) targeting DINO using Lipofectamine 2000 or 3 independent 100nM antisense oligonucleotides targeting DINO (Isis Pharmaceuticals). Large scale transfections were performed using the Amaxa Nucleofector NHDF kit. SiRNA and ASO sequences provided in Supplementary Table 3 .
Amaxa nhdf nucleofector kit
Manufactured by Lonza
The Amaxa NHDF Nucleofector kit is a laboratory equipment designed for the transfection of normal human dermal fibroblasts (NHDFs). It provides a solution-based electroporation system for efficient delivery of nucleic acids, such as plasmids or siRNAs, into NHDFs.
Automatically generated - may contain errors
Lab products found in correlation
On targetplus sirna, by Horizon Discovery (4 mentions)
Sirnas for mrnas, by Thermo Fisher Scientific (2 mentions)
Nucleofector 2 device, by Lonza (2 mentions)
Fungizone, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Hepes buffer, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Matrigel, by BD (1 mentions)
Px459, by Addgene (1 mentions)
L glutamine, by Lonza (1 mentions)
Nucleofector 2b machine, by Lonza (1 mentions)
Dmem with l glutamine, by Lonza (1 mentions)
Ham s f10 medium, by Lonza (1 mentions)
10 protocols using amaxa nhdf nucleofector kit
1
Silencing DINO in Human Fetal Lung Fibroblasts
2
Knockdown of DINO in Lung Fibroblasts
3
Knockdown of DINO in Lung Fibroblasts
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Human fetal lung fibroblasts were transfected with 3 independent 50 nM ON-TARGETPlus siRNAs (Dharmacon) or 100nM ASOs40 (link) targeting DINO using the Lipofectamine 2000 reagent. Large scale transfections were performed using the Amaxa Nucleofector NHDF kit. siRNAs for mRNAs (Ambion) were used as a pool of two. U2OS cells were infected with shRNAs targeting DINO using the pGIPZ shRNAmir lentiviral system.
4
Silencing DINO in Human Fetal Lung Fibroblasts
5
Optimizing iPSC Generation from Fibroblasts
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Early passages (between passages p3 and p5) of skin fibroblasts were cultured at low oxygen atmosphere of 4% O2/5% CO2/91% N2 and maintained in the condition for the entire reprograming period to enhance iPSC generation21 (link),26 (link). Episomal vectors carrying shRNA for p53 suppression and non-transforming MYCL, in addition to the usual reprogramming genes, POU5F1, SOX2, KLF4, and LIN28 were employed to reprogram the fibroblasts27 (link). Three micrograms of combined episomal plasmids (Addgene 27077, 27078 and 27080) were electroporated into 6 × 105 fibroblast cells by using a Nucleofector II device (Lonza) and an Amaxa NHDF Nucleofector kit (Lonza) according to the manufacturer's instructions28 (link). The electroporated cells were allowed to recover for 2 to 4 days by culturing in the above conditions. Cells (2 × 105) were placed into 100 mm dishes previously coated with Matrigel (BD Bioscience). The following day the culture medium was switched to mTeSR129 (link) (STEMCELL Technologies). Colonies emerged about 14 days post-transduction. Colonies were mechanically isolated around day 20 and expanded under feeder free conditions in mTeSR1 medium on a Matrigel coated substratum.
6
iPSC Gene Editing via Nucleofection
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When confluent, iPSCs were harvested via accutase treatment, and 1.5 × 10 (Trubetskoy et al., 2022 (link)) cells were nucleofected with 6 μg PX459 (Addgene plasmid # 62988) with a TNIK-specific sgRNA cloned into the BbsI restriction enzyme sites and 1 μL of a 100 μM custom HDR template ordered as Ultramer DNA oligos [single-stranded oligonucleotide (ssODN)] from Integrated DNA Technologies using the Amaxa NHDF nucleofector kit (Lonza, Basel, Switzerland) with program B-016. Nucleofected cells were split in equal numbers between 2 and 4 wells of a 6-well plate in mTeSR-1 media supplemented with rock inhibitor. A 48-h selection period was then started with 0.5 μg/mL puromycin. Cells were fed daily until colonies became apparent. Individual colonies were transferred into 24-well plates and allowed to grow until genotyping was subsequently carried out.
7
Porcine Fibroblast Reprogramming via Episomal Vectors
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Porcine fetal fibroblasts were reprogrammed with non-integrating episomal vectors according to previously described protocols35 (link). 3 μg of combined three episomal plasmids (Addgene #27077, 27078 and 27079) containing human POU5F1, SOX2, KLF4, LIN28, L-MYC and p53 shRNA were electroporated with a Nucleofector II device (Lonza) and Amaxa NHDF Nucleofector kit (Lonza) into 6 × 105 porcine fetal fibroblast cells derived from day 34 conceptuses36 (link). The following day, the cells were placed into three 10 cm dishes previously plated with iMEF. Next day the culture medium was switched to NHSM basal medium with FGF2 and LIF (FL condition in the first dish), FLB2i medium in the second dish and FL6i medium in the third dish. 17-day post transfection, emerged independent colonies were mechanically isolated from the dishes and expanded. A total of two iPSC lines in FL, four lines in FLB2i and eight lines in FL6i conditions were established, respectively.
8
Fibroblast Culture and Transfection
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We used primary skin fibroblasts from individuals with HS and primary skin fibroblast cell lines completely deficient of PEX1 (compound heterozygous for p.[Thr263Ilefs∗6];[Ile700Tyrfs∗42], c.[788_789del];[2097dup])19 (link) or PEX6 (homozygous for p.Gly135Aspfs∗23 [c.402del]).20 (link) Cells were cultured in DMEM with L-glutamine (Bio-Whittaker) supplemented with 10% fetal bovine serum (Bio-Whittaker), 25 mM HEPES buffer (BioWhittaker), 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies), and 250 ng/ml Fungizone (Life Technologies) in a humidified atmosphere of 5% CO2 at 37°C or 40°C. Transfections were performed with the AMAXA NHDF Nucleofector Kit (Lonza) according to the manufacturer’s instructions (program U23). The medium was changed 24 hr after transfection, and the cells were imaged 72 hr after transfection.
9
Plasmid Transfection via Electroporation
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Transfection was performed through electroporation as previously described [11 (link)]. Briefly, 1×106 cells were transfected with 4 ug plasmids (pDsRed-C1, pDsRed-C1-LA or pDsRed-C1-PG) through Amaxa NHDF Nucleofector kit (F-09376; Lonza) on a Nucleofector 2b machine (Lonza). Cells were then either seeded in 6 well plates for western blot analysis or in chamber slides for immunofluorescent staining. Doxorubicin treatment and subsequent assays were performed at 72h after transfection.
10
Functional Complementation of ACBD5 in Fibroblasts
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Primary skin fibroblasts and HeLa cells were cultured in Ham's F-10 medium (Lonza, Basel, Switzerland) or Dulbecco's modified Eagle's medium (DMEM) with L-glutamine (Bio-Whittaker) supplemented with 10% fetal bovine serum (Bio-Whittaker), 25 mM HEPES buffer (BioWhittaker), 100 U/ml penicillin, 100 mg/ml streptomycin (Life Technologies), and 250 ng/ml fungizone (Life Technologies) in a humidified atmosphere of 5% CO 2 at 37⁰C. Transfection of HeLa cells was performed in 6-well plates using the jetPRIME® DNA transfection kit (Polyplus transfection, Illkirch-Graffenstaden, France) according to the manufacturer's instructions. Transfection of skin fibroblasts was performed using the AMAXA NHDF Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer's instructions (program U23).
For functional complementation we transfected patient's skin fibroblasts with the plasmid pcDNA3.1-hsACBD5, containing full-length human ACBD5 cDNA (1.5kb). The medium was changed 24 hours after transfection. For immunofluorescence microscopy, cells were imaged 3 days after transfection. For the D3-C22:0 loading test, D3-C22:0 was added to the medium one day after transfection and fatty acid analysis was performed three days later. C26:0 lysoPC was measured in cell pellets four days after transfection.
For functional complementation we transfected patient's skin fibroblasts with the plasmid pcDNA3.1-hsACBD5, containing full-length human ACBD5 cDNA (1.5kb). The medium was changed 24 hours after transfection. For immunofluorescence microscopy, cells were imaged 3 days after transfection. For the D3-C22:0 loading test, D3-C22:0 was added to the medium one day after transfection and fatty acid analysis was performed three days later. C26:0 lysoPC was measured in cell pellets four days after transfection.
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