A total of 0.25 × 104 Cas9 TNG MKOS MEFs were mixed with 9.5 × 104 WT MEFs (129 strain) and seeded in gelatine-coated wells of 6-well plates. Cells were transduced with sgRNA lentiviruses at an MOI of 3 with 8 µg ml−1 Polybrene (Merck-Millipore) for 4 h and then reprogramming was initiated by addition of reprogramming medium. On day 14–16, whole well colony images were taken using the Celigo S Cell Cytometer (Nexcelom) and the number of Nanog-GFP+ and Nanog-GFP- colonies were counted. The images shown for illustration were stitched using Celigo S Cell Cytometer and processed using ImageJ.
Celigo s cell cytometer
Manufactured by Revvity
The Celigo S Cell Cytometer is a high-performance imaging cytometer designed for cell-based assays. It captures high-quality images and accurately quantifies various cellular parameters across multiple samples and time points.
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Lab products found in correlation
Polybrene, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
Doxycycline, by Merck Group (1 mentions)
P19arf, by Abcam (1 mentions)
Cd44 allophycocyanin apc conjugate, by Thermo Fisher Scientific (1 mentions)
E cadherin efluor 660, by Thermo Fisher Scientific (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
4 protocols using celigo s cell cytometer
1
Reprogramming of MEFs to iPSCs
2
Reprogramming of Mouse Embryonic Fibroblasts
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Nanog-GFP MEFs with or without Cas9 expression from the Rosa locus isolated from E12.5 embryos, or wild type MEFs were plated at 1.5 × 105 cells per well in a gelatin-coated 6-well plate. Twenty-four h later co-transfection of a Dox-inducible piggyBac transposon vector carrying the tetO-MKOS-ires-mOrange or tetO-STEMCCA-ires-mOrange cassette with sgRNA expression cassette, PB-CA-rtTA vector with/without carrying a P2A-linked Zfp266 cDNAs, and pCMV-hyPBase was performed using 500 ng each DNA and 6 μl of FugeneHD (Promega) as per manufacturer’s instructions20 ,46 ,47 (link). Twenty-four h later reprogramming was initiated with reprogramming medium. Medium was changed every 2 days. For colony counting, whole well colony images were taken on day 14–16 using the Celigo S Cell Cytometer (Nexcelom) and colonies were counted with ImageJ.
3
Nanog Overexpression in MEF Reprogramming
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TNG MKOS/OKMS and Nanog null MKOS MEFs were isolated from E12.5 chimeric embryos generated via morula aggregation. One-tenth of the dissociated cells were exposed to doxycycline (1,000 ng/ml) for 2 days, and mOrange expression was measured by flow cytometry to assess the proportion of Tg MEFs. For reprogramming experiments, Tg MEFs were diluted to 5% by the addition of WT MEFs and plated in a gelatinized six-well plate at 1 × 105 cells per well (5,000 Tg MEFs per well). For sorting experiments, MEFs were plated at 2 × 105 cells per gelatinized 100-mm plate with 5% Tg MEFs. All reprogramming experiments were carried out in the aforementioned ESC medium supplemented with 1.0 μg ml−1 doxycycline (Sigma) and 10 μg/ml VitC (Sigma) unless otherwise specified. Medium was changed every 2 days. Whole-well imaging and quantification of total and GFP+ colony numbers were performed with the Celigo S Cell Cytometer (Nexcelom). Overexpression of Nanog during TNG MKOS/OKMS MEF reprogramming was performed with FUW-TetO-Nanog (Addgene #40800) (Buganim et al., 2012 (link)).
4
Immunofluorescent Staining of Reprogrammed Cells
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Cells were reprogrammed in 6-well plates on 18 mm circular coverslips (Fisher Scientific) for confocal imaging, or directly on the plastic for whole well imaging, both coated with gelatin. Cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton-X in PBS for 45 minutes, blocked in 5% normal goat serum in PBS for 1 hour at room temperature, and then stained in blocking solution with primary antibody overnight at 4°C. The next day, secondary antibody was applied in blocking solution for 1 hour at room temperature. Slides where then mounted with prolong gold with or without DAPI (Life technologies). The following antibodies were used with indicated dilution: p19ARF (1:300, Abcam, Ab80), E-CADHERIN-eFluor 660 (1:300, eBioscience, #50-3249-82), CD44-allophycocyanin (APC) conjugate (1:300, eBioscience, #17-0441), pSmad3 (1:100, Cell Signaling, C25A9). For confocal microscopy, all imaging was performed with a Leica TSC SP2 and processed using Adobe Photoshop. For whole well colony and cell counting, the sistched whole well images were taken and analyzed using the Celigo S Cell Cytometer (Nexcelom).
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