Stable cell lines were generated as published [31] with minor adaptations. In short, 100.000 hiPSCs (CTL-07-II iPS) were seeded in a 12-well plate 24 h before transfection. 5 µg DNA in 2:1 ratio (PiggyBac vector : pPBase vector) were diluted in 100 µl OPTI-MEM (Thermofisher, 31985070) before adding LT-1 (Mirus/Kem-EN-Tec-Nordic, MIR 2305). The transfection mix was incubated for 15 minutes to allow formation of transfection complexes, resuspended in 900 µl medium and added onto the cells. In the first step, the synthetase plasmid was integrated, and stable cell lines were generated through selection with 1 µg/ml Puromycin (VWR, CAYM13884-100). After selection the cells were passaged in presence of apoptosis inhibitor CloneR (StemCell, #05888). In the second step the reporter plasmid (Addgene, # 140015) [32] ,was transfected, and the cells were selected with 200 µg/ml Blasticidin (Invivogen, ant-bl-10p) and 1 µg/ml Puromycin. After selection the cells were again passaged in presence of apoptosis inhibitor CloneR inhibitor (StemCell, #05888).
Cloner
Manufactured by STEMCELL
Sourced in Canada
CloneR is a device designed for automated, high-throughput single-cell cloning. It enables efficient isolation and expansion of single cells from diverse cell types, including human and mouse cells. The core function of CloneR is to facilitate the clonal expansion of individual cells for various applications, such as cell line development and genetic engineering.
Automatically generated - may contain errors
Lab products found in correlation
Y 27632, by STEMCELL (5 mentions)
Lipofectamine stem transfection reagent, by Thermo Fisher Scientific (5 mentions)
Human stem cell nucleofector kit 2, by Lonza (4 mentions)
Matrigel, by Corning (4 mentions)
Revitacell, by Thermo Fisher Scientific (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Stempro accutase, by Thermo Fisher Scientific (3 mentions)
Mtesr1, by STEMCELL (3 mentions)
Accutase, by Innovative Cell Technologies (3 mentions)
Neon transfection system, by Thermo Fisher Scientific (3 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Y 27632 hydrochloride rock inhibitor, by Cayman Chemical (2 mentions)
Mfresr, by STEMCELL (2 mentions)
Mtesr plus, by STEMCELL (2 mentions)
Pspcas9 bb 2a puro px459 v2, by Addgene (2 mentions)
Quick dna miniprep kit, by Zymo Research (2 mentions)
Lmagarose, by Bio-Techne (2 mentions)
Sybr gold, by Thermo Fisher Scientific (2 mentions)
Dmi5 microscope, by Leica camera (2 mentions)
Comet analysis software 1.3d, by Bio-Techne (2 mentions)
42 protocols using cloner
1
Stable Cell Lines Generation via PiggyBac
2
CRISPR-Mediated Gene Editing in hiPSCs
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We electroporated 1 million hiPSCs in a final volume of 100 μL R buffer containing 23 μg of ABEmax RNA, 10 μg of sgRNA, and 2 μL of electroporation enhancer. The electroporation settings were two pulses, pulse width of 20 ms, and 1100 V. Cells were seeded in a 35 mm Matrigel-coated plate with Essential 8 (E8) medium (Thermo Fisher Scientific, A1517001) containing 10 μM ROCK inhibitor, 0.1% CloneR (STEMCELL Technologies, 05888), and 175 ng/mL recombinant B18R protein (STEMCELL Technologies, 78075). At 24 h after electroporation, the medium was changed to E8 plus B18R.
3
CRISPR/Cas9 Editing of DMD-iPSCs
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The DMD-iPSCs were nucleofected with the Human Stem Cell Nucleofector® Kit 2 (LONZA, Walkersville, MD, USA) set at program B016 according to the manufacturer’s instructions. Briefly, the DMD-iPSCs were dissociated into single cells using TrypLE™ Express (Thermo Fisher Scientific) and counted. For 1.5 × 106 cells, 5 µg of CRISPR/Cas9 plasmid and donor plasmid were used, respectively, to transfect the DMD-iPSCs, then the transfected cells were cultured in mTesR1 medium with 10 µM of Y27632 (STEMCELL Technologies). Two days later, 50 µg/mL of G418 (Sigma-Aldrich) was used for screening the gene targeted cells. The cells survived from the G418 selection were counted, and 1000 cells were seeded on Matrigel-coated 6 cm dishes and cultured in mTesR1 medium with CloneR (STEMCELL Technologies) for 8 to 10 days. Then individual clones were picked, expanded and screened by PCR.
To analyze the potential off-target effect of CRISPR/Cas9, the predicted off-target sites with Cas-OFFinder [47 (link)], which is accessible athttp://www.rgenome.net . (accessed on 1 June 2021), were detected via Sanger sequencing. We isolated gDNA of hiPSCs and Rn14-iPSCs for the PCR amplification and Sanger sequencing of the top15 potential off-target sites.
To analyze the potential off-target effect of CRISPR/Cas9, the predicted off-target sites with Cas-OFFinder [47 (link)], which is accessible at
4
Generating Clonal FUCCI H9 hESC Lines
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H9 hESCs were transduced with a lentiviral vector expressing the FUCCI reporter and maintained with 300 μg/ml neomycin. After two rounds of passaging, cells were dissociated with Accutase (ThermoFisher Scientific), FACS sorted using FUCCI-driven GFP signals, and plated onto Matrigel-coated 96-well plates, which were preincubated with CloneR (Stem Cell Technologies) at a density of one cell per well. After 1 wk of media refreshing according to CloneR manufacturer’s protocol, clonal positive wells were selected with the following criteria: (1) only one colony, (2) round/compact colony shape, (3) robust GFP or mCherry expressions across the colony. Each of the clonal FUCCI lines were expanded by at least four rounds of passaging and then used for further experiments.
5
CRISPR/Cas9 Genome Editing of CCM1 in iPSCs
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The CRISPR/Cas9 genome editing protocol to generate the CCM1−/− iPSC lines used in this study was already described before [20 (link)]. In brief, AICS-0023 iPSCs were transfected with single guide RNA (sgRNA):Cas9 ribonucleoprotein complexes with the target region located in exon 10 of CCM1 (LRG_650t1, 5′-GGAGCTCCTAGACCAAAGTA-3′; Integrated DNA Technologies, Coralville, IA, USA) using Lipofectamine Stem Transfection Reagent (Thermo Fisher Scientific). IPSCs were reverse transfected following detachment with StemPro Accutase (Thermo Fisher Scientific) on growth factor reduced Matrigel-coated 24-well plates. For single-cell cloning, statistically 0.5 cells/well were plated on 96-well plates in Essential 8 Flex medium supplemented with CloneR (STEMCELL Technologies, Vancouver, BC, Canada). Genomic DNA of expanded cells was isolated and sequenced by Sanger sequencing.
6
Generation of Oligocortical Spheroids from hESCs
7
Excising Neo Cassette from TRAIL-iPSCs
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To excise the Neo cassette, 2 µg of the Cre recombinase-expressing vector pCAG-Cre-GFP was transfected using Lipofectamine (Invitrogen) in 1 × 106 integrated TRAIL-iPSCs. The next day, the transfected cells were cultured at a density of 2 × 103 cells/6-cm dish with mTeSR1 medium supplemented with 10% CloneR (STEMCELL Technologies) for 10 days; then, the standalone TRAIL-iPSC clones were picked manually and expanded for subsequent identification and use.
8
Quantifying CRISPR Editing Efficiency
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To determine editing efficiency, the cells sorted into Phire master mix (n = 4 each for DN, BFP+, BFP+/GFP+, and US) were subjected to PCR using primers flanking the edit on KCNH2 (table S1) and then sequenced via Genewiz. The editing efficiencies were determined using MoriarityLab-EditR program. Single-cell sorting was also performed on the BD FACSAria Ilu-Cell Sorter to create hiPSC clones. Specifically, BFP+/GFP+ cells were sorted as single cells into 96-well plates into a solution of CloneR (STEMCELL Technologies), mTeSR1, and 5 μM Y-27632 (STEMCELL Technologies). The medium was changed the next day with mTeSR1, and then the medium was changed every other day until visible colonies were observed. Viable colonies, once ~50% confluent, were passaged with Accutase into one well of a 24-well plate. The cells were further expanded until culture within a six-well plate, and then the clones were subjected to genotyping analysis.
9
hESC Culture and Expansion Protocol
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WA01 (H1) hESCs were obtained from WiCell at passage 46 and expanded to generate a master bank within the first 5 passages. hESCs were cultured on Matrigel (Corning, cat. no. 354277) in mTeSR1 or mTeSR Plus (STEMCELL Technologies, cat. no. 85850 and 100-0276). Passaging was performed with Accutase (Innovative Cell Technologies, cat. no. AT104-500) at split ratios from 1:6 to 1:10. hESCs were thawed in mTeSR with 10μM Y-27632 (hydrochloride) ROCK inhibitor (Cayman Chemical Company, cat. no. 10005583) and were frozen in mFreSR (STEMCELL Technologies, cat. no. 05855). Following FACS, hESCs were cultured with CloneR (STEMCELL Technologies, cat. no. 05888) per manufacturer’s instructions. Cultures were maintained at 37°C with 5% CO2. Cells were kept in sterile conditions and monitored regularly for contamination or abnormalities.
10
Generating MECP2 Indel Mutations
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The MECP2 indel mutation was generated using a previously described gene editing strategy [36 (link)]. Briefly, pSpCas9(BB)−2A-Puro (PX459) V2.0 (Addgene #62988) was cloned with an oligonucleotide designed to target MECP2 exon 3 using benchling.com CRISPR prediction tool (described in Table S2 ). Indels were introduced by transfecting 8 × 105 cells with 1.5 μg plasmid in 100 μl scale using the Neon Transfection System (ThermoFisher) with one pulse at 1500 millivolts for 30 milliseconds (~40–70% transfection efficiency). Transfected cells were plated in 2 wells of a 6-well plate with mTeSR1 media supplemented with CloneR (STEMCELL Technologies) to enhance cell survival. From D2-5, puromycin (0.5 ug/ml) was added daily to mTeSR1 media changes and surviving single colonies were grown to D12-18 before transferring to 24-well plates. Isolated clones were passaged and gDNA was harvested with a Quick DNA miniprep kit (Zymo) and PCR products were sent for Sanger sequencing by TCAG at the Hospital for Sick Children.
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