pmax GFP was obtained from Lonza (Basal, Switzerland), and pmax A3A was constructed by inserting an A3A open reading frame (NM_145699) with EcoRI/XhoI ends into an AgeI/XhoI arm of pmax GFP. A HA-A3B expression vector was obtained from NIH AIDS Reagent Program (Cat# 11090)63 (link). Plasmids were transfected into W12 cells using Fugene 6 (Promega), according to the manufacture’s instruction.
Pmaxgfp
Manufactured by Lonza
Sourced in Switzerland, Germany, United States
PmaxGFP is a plasmid vector that expresses the green fluorescent protein (GFP) under the control of a strong promoter. It can be used for a variety of applications, including cell line development, protein expression, and fluorescence-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (14 mentions)
4d nucleofector x unit, by Lonza (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Nucleofector 2 device, by Lonza (4 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (3 mentions)
Ntrna, by Horizon Discovery (3 mentions)
P3 primary cell nucleofection kit, by Lonza (3 mentions)
Effectene transfection reagent, by Qiagen (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Amaxa nucleofector, by Lonza (3 mentions)
Y 27632, by Merck Group (3 mentions)
4d nucleofector, by Lonza (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle s medium, by Euroclone (2 mentions)
Fetal bovine serum (fbs), by Euroclone (2 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
146 protocols using pmaxgfp
1
Plasmid Transfection of W12 Cells
2
Transfection of Rat E14 Spinal Cord NPCs
3
Neuronal Cell Culture Protocols
4
Electroporating NPCs to Study Ethanol Effects
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E13 mouse cortices were dissected, and the NPCs were dissociated for neurosphere suspension culture. The NPCs were cultured at 1.0 × 105 cells ml−1 in NPC medium44 (link) in the presence of 20 ng ml−1 EGF (Peprotech) and 20 ng ml−1 FGF-2 (Peprotech) for 7 days. The culture was expanded by passaging three times. For electroporation, 8 μg of control shRNA or Hsf1 shRNA15 (link), 4 μg of Hsf1 shRNA and full-length human HSF1 and 2 μg of pMAX-GFP (Amaxa) were used for 2–4 × 106 NPCs dissociated from the neurospheres. After electroporation, the NPCs were cultured with non-electroporated NPCs for 3 days to form neurospheres. The formed neurospheres with a diameter of 50-100 μm were then manually placed onto coated glass-bottom dishes (MatTek Corporation), and cultured either with 400 mM EtOH or PBS for 3 or 6 h. After fixation, migrated NPCs were counted. The cells were also immunolabelled with anti-Nestin (1:50, DSHB), anti-Ki67 (1:10, Clone B56, BD Biosciences, 550609), and anti-cleaved Caspase-3 (1:100, Cell Signaling Technology, #9654) antibodies.
5
Transwell Assay for PDGF-Induced Cell Migration
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After a 24 h serum depletion period, 1 × 106 pBSMCs were nucleofected with 1 μg pmaxGFP (Amaxa, Inc., nucleofection program A033) and ~1.6 × 105 cells seeded in each of four transwell FluoroBlok™ inserts (BD Biosciences, San Jose, CA) containing 500 μL serum-free SMCM with JNK inhibitor, MYC inhibitor or vehicle (DMSO). The transwells were placed in the corresponding wells of a companion plate containing 1 ml/well serum-free SMCM. 25 ng/ml PDGF-BB was added 60 min later to the SMCM in the bottom wells. The remaining cells were seeded in two wells of a six-well plate for confirmation of transfection efficiency. At the indicated times after adding PDGF, transwell inserts were rinsed three times with PBS for 5 min and then transferred to a glass-bottomed 24-well black plate (Greiner, Monroe, NC). GFP fluorescence signal was measured with a FLUOstar Omega microplate reader (BMG LabTech) using the bottom optic, with excitation and emission wavelengths of 485 nm and 520 nm, respectively.
6
Transfection of Chondrocytes with EZH2 Expression Vector
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EZH2 expression vector (pEZH2) was a gift from Kristian Helin (Addgene plasmid #24,230)35 (link). Plasmids were transfected in cells using nucleofection (Amaxa), according manufacturer’s protocol. Briefly, chondrocytes were harvested. Then, 4 millions of cells were mixed with 8 µg of DNA (pEZH2 or empty vector) in P3 Primary Cell 4D-Nucleofector X Solution, and placed in a 1 cm2 transfection cuvettes to be electroporated using 4D-Nucleofector X Unit. The ER-100 predefined program was used. After nucleofection, cells were plated in appropriated dishes and incubated in culture medium.
pMax-GFP (Amaxa), an expression vector for Green Fluorescent Protein, was used to evaluate transfection efficiency. The percentage of transfected cells, corresponding to green fluorescent cells, were assayed by flow cytometry. Viability was evaluated by counting adherent cells after blue trypan exclusion.
pMax-GFP (Amaxa), an expression vector for Green Fluorescent Protein, was used to evaluate transfection efficiency. The percentage of transfected cells, corresponding to green fluorescent cells, were assayed by flow cytometry. Viability was evaluated by counting adherent cells after blue trypan exclusion.
7
Endothelial SCARF1 Knockout via CRISPR-Cas9
8
Transfection of Jurkat, HEK 293T, and Primary T Cells
9
Cloning and Mutating mTOR and Rictor 3'UTRs
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The full-length 3′UTRs of mTOR and Rictor were amplified from genomic DNA (Mtor forward 5′-CTGAGGCCTGGAAAACCACGTCGTCTCC-3′, reverse 5′-TATGCTTTTAAAATTCTGATGTCATTTATTGG-3′; Rictor forward 5′-CCTCATGCTTATGACGTTTATAGCTGG-3′, reverse 5′-AAGAATTTTAAGTACATTTTATTAACAATG-3′) and cloned into pmaxGFP (Amaxa) or pRL (Promega). Mutants were made by site directed mutagenesis using the primers: Mtor let-7 site 5′-CCACTATCCTGTTtggagaACCCGTCCCTGG-3′, miR-16 site 5′-CCAACCTCCTAGCTcgacgaGAAAAGACACTGTC-3′, Rictor miR-16 site in 3,000–4,200 fragment 5′-GACCTTTTTTTTTTTTTTTTTAGTAATACcgacgatCATTTTTGGAGG-3′. A pmaxCherry plasmid was used as a transfection control.
10
Transfection and Imaging of ITSCs
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ITSCs were cultivated under standard conditions, digested with collagenase NB4 (SERVA Electrophoresis) and harvested via centrifugation. Afterwards, ITSCs were transfected with 1 μg pmax GFP (Amaxa Biosystems, Lonza Group AG, Basel, Switzerland) using Amaxa rat NSC-Nucleofector Kit (Amaxa Biosystems) and Nucleofector II device (Amaxa Biosystems) according to the manufacturer’s guidelines.
Immediate addition of 5 ml DMEM/F-12 (Sigma-Aldrich) was followed by centrifugation for 10 minutes at 300 × g. Transfected ITSCs were suspended in 2 ml standard medium supplemented with 10% BP and injected into Afc-FEP bags 2PF-0002 (Afc) using a syringe (B. Braun Melsungen AG). Cultivation was performed at 37°C, 5% CO2 and 5% O2 for 48 hours in a humidified incubator (Binder). Live imaging of GFP-ITSCs was done via optical sectioning (Z-Stack) using confocal laser scanning microscopy (excitation wavelength: 488 nm, LSM 510, Carl Zeiss, Jena, Germany), while ZEN software (Carl Zeiss) was subsequently applied for three-dimensional reconstruction.
Immediate addition of 5 ml DMEM/F-12 (Sigma-Aldrich) was followed by centrifugation for 10 minutes at 300 × g. Transfected ITSCs were suspended in 2 ml standard medium supplemented with 10% BP and injected into Afc-FEP bags 2PF-0002 (Afc) using a syringe (B. Braun Melsungen AG). Cultivation was performed at 37°C, 5% CO2 and 5% O2 for 48 hours in a humidified incubator (Binder). Live imaging of GFP-ITSCs was done via optical sectioning (Z-Stack) using confocal laser scanning microscopy (excitation wavelength: 488 nm, LSM 510, Carl Zeiss, Jena, Germany), while ZEN software (Carl Zeiss) was subsequently applied for three-dimensional reconstruction.
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