Recombinant leukemia inhibitory factor

Manufactured by Merck Group

Sourced in Germany

Recombinant leukemia inhibitory factor (rLIF) is a laboratory-produced protein that functions as a cytokine. It is used in various research applications involving cell culture and stem cell studies.

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8 protocols using recombinant leukemia inhibitory factor

Culturing Embryonic Stem Cells

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ESCs were cultured on gelatin-coated plates in standard ESC medium consisting of DMEM (Cellgro) supplemented with 15% heat-inactivated fetal bovine serum (Hyclone), 1% Glutamax (GIBCO), 1% Penicillin/Streptomycin (Cellgro), 1% nucleoside (Millipore), 0.1mM 2-mercaptoethanol (GIBCO), 1% MEM nonessential amino acids (Cellgro), and 1000U/ml recombinant leukemia inhibitory factor (Millipore).

Lu J.Y., Shao W., Chang L., Yin Y., Li T., Zhang H., Hong Y., Percharde M., Guo L., Wu Z., Liu L., Liu W., Yan P., Ramalho-Santos M., Sun Y, & Shen X. (2020). Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation. Cell reports, 30(10), 3296-3311.e5.

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Cell Culture and Transfection Protocols

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HEK 293T and BHK cells were cultured in DMEM supplemented with 10% fetal calf serum and 50 μg/ml gentamycine (PAA). MEF cells were cultured in DMEM supplemented with 15% fetal calf serum, 0.1 mM β-mercaptoethanol (Invitrogen), 2 mM l-glutamine, 1× MEM non-essential amino acids, 100 U/ml penicillin and 100 g/ml streptomycin (PAA). ESCs including J1 wt, Dnmt1^−/−, E14 wt and Uhrf1^−/− were cultured without feeder cells in gelatinized flasks as described^{33 (link)}. Culture medium was supplemented with 1 000 U/ml recombinant leukemia inhibitory factor (Millipore). The Dnmt1^−/− ESCs used in this study are homozygous for the c allele^{68 (link)}. Mouse E14 wt and Uhrf1^−/− cells have been reported before^{61 (link)}. Mouse ESCs and MEF cells were transfected with FuGENE HD (Roche), Lipofectamine® 2 000 or 3 000 reagent (Invitrogen) according to the manufacturer's instructions. HEK 293T cells and BHK cells were transfected using polyethylenimine as transfection reagent (Sigma) according to the manufacturer's instructions. Cell fixation and microscopy were carried out as described^{35 (link)}.

Qin W., Wolf P., Liu N., Link S., Smets M., Mastra F.L., Forné I., Pichler G., Hörl D., Fellinger K., Spada F., Bonapace I.M., Imhof A., Harz H, & Leonhardt H. (2015). DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination. Cell Research, 25(8), 911-929.

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Culturing Pluripotent and Cancer Cell Lines

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V6.5 (F1 hybrid of 129SvJae/C57BL/6) mESCs and ZHBTc4 cells were maintained and expanded on gelatin-coated (Millipore) plates in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) containing 15% FBS (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 2 mM GlutaMAX™-I (GIBCO), 0.1 mM MEM nonessential amino acids (NEAA; GIBCO), 1 mM sodium pyruvate (GIBCO), 1000 U/ml recombinant leukemia inhibitory factor (Millipore), and 30 U/ml penicillin/streptomycin (GIBCO). Approximately 200k cells were seeded in each 6-well plate (9.5 cm²/well; Corning). The media were changed daily, and mESCs were dissociated and expanded every 2 days.
MCF7 (human epithelial breast carcinoma) cells were cultured in DMEM (GIBCO) supplemented with 10% FBS (GIBCO) and 100 U/ml penicillin/streptomycin (GIBCO). Cells were seeded in each 6-well plate (9.5 cm²/well; Corning) and sub-cultured upon reaching 80–90% confluence.
HEK293T cells were cultured in DMEM (GIBCO) supplemented with 10% FBS (GIBCO) and 100 U/ml penicillin/streptomycin (GIBCO). Cells were seeded in the plate (Corning) and sub-cultured upon reaching 80–90% confluence.

Xiao Z., Cheng G., Jiao Y., Pan C., Li R., Jia D., Zhu J., Wu C., Zheng M, & Jia J. (2018). Holo-Seq: single-cell sequencing of holo-transcriptome. Genome Biology, 19, 163.

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Culturing Embryonic Stem Cells

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Maintenance of Cell Lines and Organoids

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The mES cells (original from Novus Biologicals, NBP1-41162) were provided by H. Deng, Peking University, and maintained in this laboratory. The HGC27 cell line (original from ECACC, CVCL_1279) and SNU16 cell line (original from ATCC, CRL-5974) were provided by S. Shu, Peking University Cancer Hospital & Institute, and maintained in this laboratory. The human K562 cell line (original from ATCC, CCL-243) was maintained in this laboratory. All cells were cultured at 37 °C with 5% CO 2 . K562 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma). SNU16 cells were cultured in DMEM (Hyclone) supplemented with 10% FBS. HGC27 cells were cultured in RPMI 1640 (Gibco) supplemented with 20% FBS. The mES cells were cultured in DMEM (Hyclone) supplemented with 15% FBS, 1% Glutamax (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 1% MEM nonessential amino acids (Cellgro), 1% nucleoside (Millipore) and 1,000 U ml -1 recombinant leukemia inhibitory factor (Millipore). CRC organoids were established as described previously and cultured in CRC organoid medium 55 .

Dong C., Meng X., Zhang T., Guo Z., Liu Y., Wu P., Chen S., Zhou F., Ma Y., Xiong H., Shu S, & He A. (2024). Single-cell EpiChem jointly measures drug-chromatin binding and multimodal epigenome. Nature methods.

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Culturing Murine ESCs, K562, and H1 hESCs

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Wild-type V6.5 murine ESCs were cultured on 0.1% gelatin-coated plates in ESC Dulbecco’s modified Eagle’s medium culture medium containing 15% fetal bovine serum (Sigma-Aldrich), 1% penicillin/streptomycin (Hyclone), 1% GlutaMAX (Hyclone), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 1% minimum essential medium (MEM) nonessential amino acids (CellGro), 1% nucleoside (Millipore), and recombinant leukemia inhibitory factor (1000 U/ml; Millipore). V6.5 cells were digested with 0.1% trypsin (Hyclone) at 37°C for 3 min to harvest single cells. K562 and Kasumi-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (Sigma-Aldrich), 1% penicillin/streptomycin (Hyclone), and 1% GlutaMAX (Hyclone). K562 and Kasumi-1 cells were harvested by centrifugation for 2 min at 600g. H1 hESCs were cultured in Matrigel (Corning)–coated plates using hESC culture medium mTeSR1. H1 cells were digested with Accutase (Thermo Fisher Scientific) at 37°C for 3 min to harvest single cells.

Xiong H., Wang Q., Li C.C, & He A. (2024). Single-cell joint profiling of multiple epigenetic proteins and gene transcription. Science Advances, 10(1), eadi3664.

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Genetic Modification of mESCs

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CV19 ES cells (passage 13 [129Sv × C57BL/6J]) were expanded in HEPES‐buffered Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum (PAA), non‐essential amino acids, L‐glutamine, β‐mercaptoethanol, 1,000 U of recombinant leukemia inhibitory factor (MERCK Millipore, Germany) per ml, and antibiotics (penicillin [100 U/ml] and streptomycin [100 μg/ml]). For electroporation, 2 × 10⁷ cells were resuspended in 0.8 ml Capecchi buffer (20 mM HEPES [pH 7.4], 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM dextrose, 0.1 mM β‐mercaptoethanol. The targeting vector pTMEM16a_targ was linearized with NotI, and 55 μg of DNA was electroporated at 25 μF and 400V in 0.8‐mm electroporation cuvettes (Gene Pulser; Bio‐Rad). After electroporation, cells were cultivated for 10 min at room temperature and plated onto 10 culture dishes containing a gamma‐irradiated monolayer of mouse primary G418‐resistant fibroblast feeder cells. Thirty‐two hours later, 350 µg of G418 (Invitrogen) per ml and 0.2 μM 2’‐deoxy‐2’‐fluoro‐β‐D‐arabinofuranosyl‐5‐iodouracil (FIAU; Moravek Biochemicals and Radiochemicals) were added to the culture medium. The medium was replaced every day, and colonies were picked and analyzed 8 days after plating.

Matchkov V.V., Black Joergensen H., Kamaev D., Hoegh Jensen A., Beck H.C., Skryabin B.V, & Aalkjaer C. (2020). A paradoxical increase of force development in saphenous and tail arteries from heterozygous ANO1 knockout mice. Physiological Reports, 8(22), e14645.

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Targeted Gene Editing in Mouse Embryonic Stem Cells

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CV19 embryonic stem (ES) cells [passage 13 (129Sv ϫ C57BL/ 6J)] were expanded in HEPES-buffered Dulbecco's modified Eagle's medium, supplemented with 15% fetal bovine serum (PAA), nonessential amino acids, L-glutamine, ␤-mercaptoethanol, 1,000 U/ml recombinant leukemia inhibitory factor (Merck Millipore), and antibiotics [penicillin (100 U/ml) and streptomycin (100 g/ml)]. For electroporation, 2 ϫ 10 7 cells were resuspended in 0.8 ml Capecchi buffer [20 mM HEPES (pH 7.4), 173 mM NaCl, 5 mM KCl, 0.7 mM Na 2HPO4, 6 mM dextrose, and 0.1 mM ␤-mercaptoethanol] (37). The targeting vector pSERCA VAC14 L156R (100 g) was linearized with SgfI and electroporated together with the ROSA26_ZFN (L6 and R4) coding plasmids (25) (25 g each) at 25 F and 400 V in 0.8 mm electroporation cuvettes (Gene Pulser; Bio-Rad). After electroporation, cells were cultivated for 10 min at room temperature and plated onto 10, 100-mm diameter culture dishes containing a gamma-irradiated monolayer of mouse primary G418-resistant fibroblast feeder cells. Thirty-two hours later, 350 g/ml G418 (Invitrogen) were added to the culture medium. The medium was replaced every day, and colonies were picked and analyzed 8 days after plating.

Michgehl U., Skryabin B.V., Bayraktar S., Vollenbröker B., Ciarimboli G., Heitplatz B., Van Marck V., Gröne H.J., Pavenstädt H, & Weide T. (2018). Nephron-specific knockin of the PIKfyve-binding-deficient Vac14^L156R mutant results in albuminuria and mesangial expansion. American journal of physiology. Renal physiology, 315(5).

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