Sc 5279

Manufactured by R&D Systems

Sc-5279 is a laboratory equipment product offered by R&D Systems. It is a device used for scientific applications, though the core function is not specified in detail.

Automatically generated - may contain errors

Lab products found in correlation

Ab8978, by Abcam (1 mentions) Af2085, by R&D Systems (1 mentions) Af2400, by Abcam (1 mentions) Mab1924, by R&D Systems (1 mentions) Af1700, by Cell Signaling Technology (1 mentions) Af2864, by Thermo Fisher Scientific (1 mentions) Mab4360, by Merck Group (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Accutase, by Merck Group (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Gene pulser xcell electroporation system, by Bio-Rad (1 mentions) Af2018, by R&D Systems (1 mentions)

4 protocols using sc 5279

Immunofluorescence Analysis of Pluripotency and Lineage Markers

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OCT4: Santa Cruz Biotechnology (sc-5279), 1:200

BRA: R&D Systems (MAB20851-100), 1:500

BRA: R&D Systems (AF2085), 1:300

GATA6: R&D Systems (AF1700), 1:200

GATA6: Cell Signaling (D61E4), 1:200

GATA3: Thermo Fisher Scientific (14-9966-82), 1:100

SOX17: R&D Systems (MAB1924), 1:200

HAND1: R&D Systems (AF3168-SP), 1:200

KRT7: Abcam (ab209600), 1:100

ISL1: DSHB (39.4D5), 1:200

MIXL1: Sigma Prestige antibody (HPA005662), 1:200

SUSD2: Miltenyi Biotec (130-106-401), 1:100

SSEA4: Miltenyi Biotec (130-122-958), 1:100

KLF17: Atlas Antibodies (HPA024629), 1:200

SOX17: R&D Systems (AF2864), 1:200

GATA4: Thermo Fisher Scientific (14-9980-82), 1:100

FOXA2: Novus Biologicals (AF2400), 1:200

NR2F2: Abcam [EPR18442] (ab211776), 1:100

CD24: eBioscience (A5-2H10), 1:100

Vimentin: Abcam (ab8978), 1:200

De Santis R., Rice E., Croft G., Yang M., Rosado-Olivieri E.A, & Brivanlou A.H. (2023). The emergence of human gastrulation upon in vitro attachment. Stem Cell Reports, 19(1), 41-53.

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Generation of Human Induced Pluripotent Stem Cells

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Human control fibroblast was purchased from the Coriell Cell Repository. Human fibroblasts (GM23967, male, 52 years old) were cultured in human cell-culture medium containing DMEM, 10% fetal bovine serum, 1% non-essential amino acids (Gibco), 0.1% β-mercaptoethanol (Gibco), and 1% penicillin/streptomycin (Gibco). To generate hiPSCs from fibroblasts, we infected cells at passage 6 with a lentivirus construct harboring Oct4, Sox2, C-myc, and Klf4 twice in 2 days. The hiPSC line was cultured in human embryonic stem cell growth medium (CEFO, Seoul, South Korea) on Matrigel at 37°C and 5% CO₂. For the transduction of donor and CRISPR/Cas9 vectors, we treated hiPSCs with Accutase (A6964, Sigma) and transfected them using the Gene Pulser Xcell Electroporation System (Bio-Rad). These cells were seeded on Matrigel-coated plates, and human embryonic stem cell growth medium was changed every day. The hiPSCs were immunostained using primary antibodies against OCT4 (Santa Cruz Biotechnology, sc-5279), SOX2 (R&D Systems, MAB2018), NANOG (Bethyl Laboratories, A300-397a), SSEA1 (Santa Cruz, sc-21702), and TRA 1–60 (Millipore, MAB4360), and appropriate fluorescent secondary antibodies (Invitrogen).

Kim H., Park H.J., Choi H., Chang Y., Park H., Shin J., Kim J., Lengner C.J., Lee Y.K, & Kim J. (2019). Modeling G2019S-LRRK2 Sporadic Parkinson's Disease in 3D Midbrain Organoids. Stem Cell Reports, 12(3), 518-531.

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Immunofluorescence Assay for Pluripotency Markers

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Cells in 12-well plates were fixed in PBS supplemented with 4% paraformaldehyde for 20 min at room temperature. The cells were then permeabilized and blocked using 0.2% Triton X-100, 0.1% Tween-20 and 3% donkey serum in PBS for 30 min. Primary antibody against human OCT4 (1:500, Santa Cruz, sc-5279), human SOX2 (1:500, R&D Systems, AF2018), and human NANOG (1:250, goat polyclonal, R&D Systems, AF1997) was diluted in 0.2% Triton X-100 in PBS and incubated with the samples overnight at 4 °C. The cells were treated with an appropriate Molecular Probes Alexa Fluor® dye conjugated secondary antibodies (Donkey-α-Mouse Alexa Fluor® Plus 594 Cat# A32744 and Donkey-α-Goat Alexa Fluor® Plus 488 Cat# A32814, both 1:500, Invitrogen) and then incubated for 1 h. The nuclei were stained with DAPI for 10 min.

Huang X., Park K.M., Gontarz P., Zhang B., Pan J., McKenzie Z., Fischer L.A., Dong C., Dietmann S., Xing X., Shliaha P.V., Yang J., Li D., Ding J., Lungjangwa T., Mitalipova M., Khan S.A., Imsoonthornruksa S., Jensen N., Wang T., Kadoch C., Jaenisch R., Wang J, & Theunissen T.W. (2021). OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human. Nature Communications, 12, 5123.

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Induced Pluripotent Stem Cell Generation

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Fibroblasts were seeded at 150,000 cells per 12-well in 5 wells per line (resulting in 5 PSC sub-clones) and transduced with 4 CytoTune Sendai viruses (Life Technologies, A1378002) encoding Oct-4, Sox2, Klf4 and c-Myc each at an MOI of 3¹⁶. Cells were fed daily with fibroblast medium. At day 7, the cells were dissociated using trypsin-EDTA (Gibco-Life Technologies, 25300-054) and replated onto 10-cm MEF (GlobalStem, GSC-6105M)-coated plates in HES medium supplemented with 0.5 mM VPA (Tocris Bioscience, 2815), as described in Zeltner et al,¹⁸. Cells were fed daily with HES medium/VPA until day 14, then with HES medium only. Between day 20 and 40 PSC colonies were manually picked, expanded and frozen. PSCs were fed daily with HES medium and passaged manually or with trypsin-EDTA¹⁸ approximately once per week onto MEFs. PSC cultures were routinely tested for mycoplasma contamination every other week. For the characterization of PSC lines, the cells were immuno-stained with Oct-4 (mIgG2b, Santa Cruz, sc-5279), Nanog (goat, R&D, AF1997) and Sendai virus (rabbit, MBL International, PD029). IKBKAP splicing in PSCs was conducted as described for fibroblasts. The karyotypes of the PSC clones were normal, except at high passages in S3 and M2.

Zeltner N., Fattahi F., Dubois N.C., Saurat N., Lafaille F., Shang L., Zimmer B., Tchieu J., Soliman M.A., Lee G., Casanova J.L, & Studer L. (2016). Capturing the biology of mild versus severe disease in a pluripotent stem cell-based model of Familial Dysautonomia. Nature medicine, 22(12), 1421-1427.

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