NCCIT, HEK293 cells were obtained from ATCC. NCCIT cells were maintained in 10% FBS (Omega), 1× Glutamax-I supplement (100× stock, Invitrogen), 1xNon-essential amino acids (100x stock, invitrogen), and basal media RPMI 1640 (Hyclone). HEK293 cells were maintained in 10% FBS, 1x NEAA, 1x glutamax in DMEM-high glucose (Hyclone). hESCs (H9 line, Wi-Cell) were used at passage 60–67 and were expanded in feeder-free, serum-free medium, mTESR-1 from StemCell technologies. HESC HSF-1 (male) and HSF-8 (male) hESC were used at passage 20–28, cultured as described above and their characterization is described elsewhere31 . Cells were passaged 1:7 every 5–6 days by incubation with accutase (Invitrogen) and resultant small cell clusters (50–200 cells) were subsequently re-plated on tissue culture dishes coated overnight with growth-factor-reduced matrigel (BD Biosciences). ELF1 naïve hESC were obtained from Dr. Carol Ware and cultured as previously described14 (link), with 10ng/mL human recombinant LIF (R&D). Cell cultures were routinely tested and found negative for mycoplasma infection (MycoAlert, Lonza).
Recombinant human lif
Manufactured by R&D Systems
Recombinant human LIF is a cytokine that promotes the self-renewal of undifferentiated embryonic stem cells. It is produced in a human cell expression system.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax 1 supplement, by Thermo Fisher Scientific (2 mentions)
H9 line, by WiCell (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Growth factor reduced matrigel, by BD (2 mentions)
Mycoalert, by Lonza (2 mentions)
Mtesr1, by STEMCELL (2 mentions)
Human tgf β1, by R&D Systems (2 mentions)
Human oncostatin m, by R&D Systems (2 mentions)
Mouse lif, by Miltenyi Biotec (2 mentions)
Mouse oncostatin m, by R&D Systems (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Human il 6, by R&D Systems (1 mentions)
Human il 6rα, by R&D Systems (1 mentions)
Human osm, by R&D Systems (1 mentions)
7 protocols using recombinant human lif
1
Maintenance and Culture of Cell Lines
2
Differentiation of E14 ESCs to EBs and Monolayers
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E14 ESCs were a gift from Professor Austin Smith (Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK). E14 cells and TIAR RNAi E14 cells were maintained on gelatin-coated dishes in the absence of feeder cells in GMEM (Sigma, St. Louis, MO), supplemented with 2 mM glutamine (Gibco, Grand Island, NY), 0.001% β-mercaptoethanol (Gibco), 1× nonessential amino acids (Gibco), 1% sodium pyruvate (Gibco), 10% fetal bovine serum (FBS) (Gibco), and 2,000 units/mL human recombinant LIF (R & D Systems, Minneapolis, MN).
For differentiation of E14 cells into EBs, TIAR RNAi E14 cells (4 × 105 cells per mL) were plated in uncoated Petri dishes in medium containing GMEM/10% FBS without LIF. Cells grew in suspension and formed EBs. The expression of markers specific to the three germ layers was assayed via RT-PCR or qRT-PCR.
For the first stage of monolayer differentiation, E14 cells and TIAR RNAi E14 cells (1 × 104 cells per mL) were cultured on collagen type-IV in GMDM medium, 10% FBS, without LIF for 4 days. In the second stage, cells were cultured on collagen type-IV in GMDM medium, 10% FBS, without LIF and containing vascular endothelial growth factor (VEGF) (PeproTech) for another 4 days.
For differentiation of E14 cells into EBs, TIAR RNAi E14 cells (4 × 105 cells per mL) were plated in uncoated Petri dishes in medium containing GMEM/10% FBS without LIF. Cells grew in suspension and formed EBs. The expression of markers specific to the three germ layers was assayed via RT-PCR or qRT-PCR.
For the first stage of monolayer differentiation, E14 cells and TIAR RNAi E14 cells (1 × 104 cells per mL) were cultured on collagen type-IV in GMDM medium, 10% FBS, without LIF for 4 days. In the second stage, cells were cultured on collagen type-IV in GMDM medium, 10% FBS, without LIF and containing vascular endothelial growth factor (VEGF) (PeproTech) for another 4 days.
3
Maintenance and Culture of Cell Lines
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NCCIT, HEK293 cells were obtained from ATCC. NCCIT cells were maintained in 10% FBS (Omega), 1× Glutamax-I supplement (100× stock, Invitrogen), 1xNon-essential amino acids (100x stock, invitrogen), and basal media RPMI 1640 (Hyclone). HEK293 cells were maintained in 10% FBS, 1x NEAA, 1x glutamax in DMEM-high glucose (Hyclone). hESCs (H9 line, Wi-Cell) were used at passage 60–67 and were expanded in feeder-free, serum-free medium, mTESR-1 from StemCell technologies. HESC HSF-1 (male) and HSF-8 (male) hESC were used at passage 20–28, cultured as described above and their characterization is described elsewhere31 . Cells were passaged 1:7 every 5–6 days by incubation with accutase (Invitrogen) and resultant small cell clusters (50–200 cells) were subsequently re-plated on tissue culture dishes coated overnight with growth-factor-reduced matrigel (BD Biosciences). ELF1 naïve hESC were obtained from Dr. Carol Ware and cultured as previously described14 (link), with 10ng/mL human recombinant LIF (R&D). Cell cultures were routinely tested and found negative for mycoplasma infection (MycoAlert, Lonza).
4
Breast Cancer Cytokine Stimulation Assay
5
Cytokine Reconstitution and Cell Treatment
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Recombinant human LIF (R&D Systems), human oncostatin M (R&D Systems), mouse LIF (Miltenyi Biotec), mouse oncostatin M (R&D Systems), and human TGF-β1 (R&D Systems) were reconstituted in PBS + 0.1% bovine serum albumin (BSA) at 10–25μg/ml and aliquoted for storage at −80°C. For all experiments, mouse recombinant proteins were used on mouse cell lines, and human recombinant proteins were used on human cell lines, with the exception of human TGF-β1, which was used on all cell lines since TGF-β1 maintains approximately 99% sequence homology between human and mouse species63 (link). Prior to cytokine treatment cells were serum starved in 2% FBS overnight and cytokine treatment was made up in media containing 2% FBS.
6
Cytokine Reconstitution and Cell Treatment
7
Evaluating LIF/LIFR Binding Profiles using SPR
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