Revitacell

Manufactured by Thermo Fisher Scientific

Sourced in United States

RevitaCell is a cell culture supplement designed to maintain and support the viability of cells in culture. It is a serum-free, chemically defined formulation that provides essential nutrients and growth factors to promote cell growth and proliferation. RevitaCell is suitable for use with a variety of cell types, including stem cells, primary cells, and established cell lines.

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Lab products found in correlation

62 protocols using revitacell

Measuring YAP/TAZ Activity in Stiffness Sensing

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The TC28a2 Yes-associated protein and TAZ transcriptional coactivator with PDZ-binding motif (YAP/TAZ)_TEAD reporter cell line^{29 (link)} was used as a reporter for YAP/TAZ activity and extracellular matrix (ECM) stiffness.
Rho/Rho-associated protein kinase (ROCK) signalling pathways participate in stiffness sensing through stress fibre formation.^{29 (link)} ROCK inhibitors have been shown to decrease cell tension.^{30 (link)} Therefore, Revitacell (#A2644501, ThermoFisher) containing a specific ROCK inhibitor complex different from Y-27632 was used as a positive control in this experiment.
A total of 10,000 reporter cells were seeded into each well of a black walled 96 well plate and incubated overnight, following which cells were serum starved for a further 24 hours. Then us-GO (5 μg ml⁻¹), Revitacell (#A2644501, Gibco) (1 : 100), TGFβ-3 (10 ng ml⁻¹) was applied to cells by diluting in serum-free medium. After 4 hours Nanoglo live reagent (Promega) was applied to cells and luminescence was read using the GloMax-Multi Detection system (Promega). The assay output, relative luminesce units (RLU) are a measure of YAP/TAZ activity through interaction with TEAD1.

Ogene L., Woods S., Hetmanski J., Lozano N., Karakasidi A., Caswell P.T., Kostarelos K., Domingos M.A., Vranic S, & Kimber S.J. (2024). Graphene oxide activates canonical TGFβ signalling in a human chondrocyte cell line via increased plasma membrane tension. Nanoscale, 16(11), 5653-5664.

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Quantifying Cell Proliferation via MTT Assay

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Cell proliferation was assessed using the Abcam MTT Cell Proliferation Assay Kit (Abcam) according to manufacturer’s instructions. In brief, 5 x 10³ cells/well were seeded in 96-well tissue culture plates in triplicate in TeSR-E8 medium supplemented with Revitacell (Gibco) (10μl Revitacell per 1ml medium) and incubated for 48h. To quantify metabolically active cells, medium was replaced with a 50:50 mix of MTT reagent and TeSR-E8 supplemented with Revitacell and cells incubated for 3h at 37°C. MTT solvent was added to each well and the plate shaken in the dark on an orbital shaker for 15min. Absorbance at 595nm was immediately assessed spectrophotometrically.

Wood K.A., Rowlands C.F., Thomas H.B., Woods S., O’Flaherty J., Douzgou S., Kimber S.J., Newman W.G, & O’Keefe R.T. (2020). Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells. PLoS ONE, 15(7), e0233582.

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CRISPR-Cas9 Genome Editing of KANSL1 in iPSCs

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We performed CRISPR-Cas9 genome editing on C₁ following the protocol from Ran et al. [88 (link)]. First sgRNA targeting exon 2 of KANSL1 was cloned into the targeting vector (pX459v2; Addgene, 62988; deposited by Feng Zhang). Successful cloning was validated by PCR. Single iPSCs were then nucleofected with the pX459v2 plasmid coding for the Cas9 protein and the sgRNA using P3 primary Cell 4D nucleofector kit (Lonza, V4XP-302). After nucleofection cells were plated on a 6-well plate in E8 flex medium (Gibco, A2858501) supplemented with RevitaCell (Gibco, A2644501).24 h after nucleofection medium was refreshed and supplemented with puromycin (1 µg/ml), RevitaCell was removed. After overnight incubation medium was refreshed with E8 flex to remove dead cells and stop the selection. The culture was maintained until colonies were large enough to be picked. By means of Sanger sequencing we checked for heterozygous loss-of-function mutations. Positive colonies were re-plated as single cells to ensure clonal expansion of iPSCs positive for the selected mutation. KANSL1 sgRNA oligos:
5ʹ- CACCGGAGCCCGTTTTCCCCCATTG-3ʹ;
3ʹ-CCTCGGGCAAAAGGGGGTAACCAAA-5ʹ

Linda K., Lewerissa E.I., Verboven A.H., Gabriele M., Frega M., Klein Gunnewiek T.M., Devilee L., Ulferts E., Hommersom M., Oudakker A., Schoenmaker C., van Bokhoven H., Schubert D., Testa G., Koolen D.A., de Vries B.B, & Nadif Kasri N. (2021). Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders. Autophagy, 18(2), 423-442.

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MTT Assay for Cell Proliferation

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Cell proliferation was assessed using the Abcam MTT Cell Proliferation Assay Kit (Abcam)
according to manufacturer's instructions. In brief, 5 x 10 3 cells/well were seeded in 96-well tissue culture plates in triplicate in TeSR-E8 medium supplemented with Revitacell (Gibco) (10l Revitacell per 1ml medium) and incubated for 48h. To quantify metabolically active cells, medium was replaced with a 50:50 mix of MTT reagent and TeSR-E8 supplemented with Revitacell and cells incubated for 3h at 37C. MTT solvent was added to each well and the plate shaken in the dark on an orbital shaker for 15min. Absorbance at 595nm was immediately assessed spectrophotometrically.

Wood K., Rowlands C.F., Thomas H.B., Woods S.P., O’Flaherty J., Douzgou S., Kimber S.J., Newman W.G., & O’Keefe R.T. (2020). Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells.

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Differentiation of iPSCs to Cardiomyocytes

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Cell reprogramming into iPSC lines was previously described by Rovina et al. [15 (link)]. Briefly, iPSCs were seeded in Matrigel (Corning, 356,230) coated 6 well plates in StemFlex medium (ThermoFisher, A3349401) supplemented with RevitaCell (ThermoFisher, A2644501). On Day 0, medium was replaced with BPEL medium and cells were treated with 6 mM CHIR99021 (Bertin bioreagent, 13,122), 100 µg/ml Activin A (MiltenyiBiotec, 130–115-008), and 100 µg/ml BMP4 (R&D Systems, 5020-BP). On Day 3, cells were treated with 10 mM XAV939 (R&D Systems,3748), 100 µg/ml BMP4 and 2 mM Retinoic acid (Sigma Aldrich, R2625). Medium is changed on Day6 with BPEL + 30 ng/ml BMP4 + 1 µM Retinoic acid. After 3 days, cells were replated on fibronectin coated plates with BPEL medium supplemented with 20 mM SB 431542 (R&D Systems,161).

Soussi S., Savchenko L., Rovina D., Iacovoni J.S., Gottinger A., Vialettes M., Pioner J.M., Farini A., Mallia S., Rabino M., Pompilio G., Parini A., Lairez O., Gowran A, & Pizzinat N. (2023). IPSC derived cardiac fibroblasts of DMD patients show compromised actin microfilaments, metabolic shift and pro-fibrotic phenotype. Biology Direct, 18, 41.

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Genome-edited hiPSC line expressing AP2-tagRFP-T

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WTC-10 human induced pluripotent stem cells (hiPSCs) were obtained from the lab of Bruce Conklin and genome edited using TALENs to endogenously express AP2-tagRFP-T at one allele at an internal loop of the µ2 subunit (Hong et al., 2015 (link)). We grew these cells on matrigel (hESC-Qualified Matrix, Corning) (80 µg/mL, 1 mL/well) in StemFlex (Thermo Fisher) with Penicillin/Streptomycin (Thermo Fisher), and passaged with Gentle Cell Dissociation reagent (EDTA-based clump passaging; StemCell Technologies). Parental and genome-edited cells were tested for mycoplasma and authenticated by STR profiling. For single-cell applications (genome editing, flow cytometry, transfections) we trypsinized the cells with the recombinant trypsin TrypLE Select (Thermo Fisher) and grew the cells in StemFlex supplemented with the specific rho kinase inhibitor RevitaCell (Thermo Fisher).

Akamatsu M., Vasan R., Serwas D., Ferrin M.A., Rangamani P, & Drubin D.G. (2020). Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis. eLife, 9, e49840.

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hiPSC-CM Culture and Maintenance

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hiPSC-CMs were thawed and maintained in Medium C (Ncardia) or a modified BPEL medium (mBEL) as previously described.^{33 (link),34 (link)} Thawed hiPSC-CMs were replated on Matrigel-coated cell culture plates in medium C or mBEL supplemented with RevitaCell (1:100 dilution; Thermo-Fisher) at a density of 1.6-1.9 × 10⁵ cells/cm² for 96-well plates or 1.3-1.6 × 10⁵ cells/cm² for all other formats. The medium was refreshed 24 h later, and subsequently every 2-3 days thereafter.

Yiangou L., Blanch-Asensio A., de Korte T., Miller D.C., van Meer B.J., Mol M.P., van den Brink L., Brandão K.O., Mummery C.L, & Davis R.P. (2022). Optogenetic Reporters Delivered as mRNA Facilitate Repeatable Action Potential and Calcium Handling Assessment in Human iPSC-Derived Cardiomyocytes. Stem Cells, 40(7), 655-668.

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Human Induced Pluripotent Stem Cell Maintenance

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The established hiPSC lines were cultured in feeder-free conditions on Vitronectin (VTN-N) (Thermo Fisher Scientific, Cat#A14700) coated plates in E8 medium (Thermo Fisher Scientific, Cat#A1517001) and passaged with Versene (Thermo Fisher Scientific, Cat#15040066) in E8 medium supplemented with RevitaCell™ (Thermo Fisher Scientific, Cat#A2644501) in 5% CO2 and 5% O2 at 37°C. For NRF1 silencing experiment iPSCs have been transfected with three independent siRNAs (50 nM; Supplementary Table S13) using RNAiMAX Lipofectamine (Thermo Scientific). Cells were incubated in E8 medium for 2 days after transfection before collection for RNA extraction.

Astro V., Alowaysi M., Fiacco E., Saera-Vila A., Cardona-Londoño K.J., Aiese Cigliano R, & Adamo A. (2022). Pseudoautosomal Region 1 Overdosage Affects the Global Transcriptome in iPSCs From Patients With Klinefelter Syndrome and High-Grade X Chromosome Aneuploidies. Frontiers in Cell and Developmental Biology, 9, 801597.

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Neutral Comet Assay for hPSCs

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hPSCs were subjected to neutral comet assays and performed according to the manufacturer’s protocol (Trevigen, #4260–096). Cells were dissociated with Accutase for 10 min at 37°C. To ensure single-cell dissociation, the cell suspension was gently pipetted up and down washed four times with PBS. Cells were then pelleted, resuspended, filtered through 15-μm cell strainer and seeded onto the 96-well Comet chip (5,000 cells/well) containing E8 Medium supplemented with DMSO, Y-27632, CloneR (STEMCELL Technologies), RevitaCell (Thermo Fisher Scientific), SMC4 (BioVision) or CEPT. Cells were allowed to settle and were incubated for 6 h. Subsequently, the 96-well comet chip was rinsed, overlayed with LMAgarose (Trevigen) and allowed to set for 3 min. Comet chip was placed in a lysis solution for 2 h at 4°C, equilibrated in neutral solution and subjected to electrophoresis at 4°C for 50 min at 22 V in neutral solution. Comet chip was stained overnight at 4°C in 0.2X SYBR Gold (Thermo Fisher Scientific). Comets were visualized using a Leica DMi5 microscope using the appropriate filters. Comets were analyzed using the Comet Analysis Software 1.3d (Trevigen).

Chen Y., Tristan C.A., Chen L., Jovanovic V.M., Malley C., Chu P.H., Ryu S., Deng T., Ormanoglu P., Tao D., Fang Y., Slamecka J., Hong H., LeClair C.A., Michael S., Austin C.P., Simeonov A, & Singeç I. (2021). A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells. Nature methods, 18(5), 528-541.

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Efficient Genome Editing in hESCs

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H1 hESCs were pretreated with Revitacell (Thermo Fisher Scientific, cat. no. A2644501) for 1h, then nucleofected (Lonza Nucleofector 4D, program CA-137) with a Cas9 construct (modified version of pMJ915, a gift from Chris Jeans) and up to three gRNA-encoding vectors to target each gene. The pool of transfectants was then subjected to MiSeq analysis (Illumina) to estimate the frequency of frameshift mutations in each targeted gene. Individual colonies were picked and screened via Miseq to identify clones with frameshift mutations, which were then single cell sorted. Another round of Miseq was performed to verify the mutations and confirm the absence of mosaicism. Clones were karyotyped by Cell Line Genetics. Clones with normal karyotypes were expanded and frozen down within the first five passages, which were used to generate subsequent lines. The gRNAs and primers used for each targeted gene are listed in Table S5. See the Supplemental experimental procedures for the TP53 mutation correction and whole exome sequencing.

Pizzato H.A., Alonso-Guallart P., Woods J., Connelly J.P., Fehniger T.A., Atkinson J.P., Pruett-Miller S.M., Monsma FJ J.r, & Bhattacharya D. (2024). Engineering human pluripotent stem cell lines to evade xenogeneic transplantation barriers. Stem Cell Reports, 19(2), 299-313.

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