Human thrombopoietin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Human thrombopoietin is a recombinant protein that stimulates the production and differentiation of megakaryocytes, which are the precursor cells for platelets. It is a key regulator of platelet production in the body.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Human thrombopoietin (TPO) Human thrombopoietin (hTPO) Thrombopoietin

6 protocols using human thrombopoietin

Establishing and Maintaining AML and HEK293T Cell Lines

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

AML cell lines and HEK293T cells were maintained under standard conditions with up to 0.01% DMSO. Cell line identity was verified using the Multiplex Cell Authentication Test (Multiplexion, Friedrichshafen, Germany) or the Human Cell Line Authentication Service (Eurofins Genomics GmbH, Ebertsberg, Germany). All cell lines were routinely tested for mycoplasma contamination. AML PDX cells were isolated from the spleen of leukemic mice as described previously^{56 (link)} and cultured in StemPro34 SFM (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with provided nutrient supplement, 2% FBS, 1% penicillin/streptomycin and human FLT3 ligand, human thrombopoietin, human interleukin 3, and human stem cell factor (PeproTech, Princeton, New Jersey, USA; 10 ng/mL each). Murine cell lines were a gift from Stephen M. Sykes and were generated and cultured as described previously^{57 (link)} (liquid culture) or in methylcellulose medium (MethoCult GF M3434, Stem Cell Technologies, Vancouver, Canada). Palbociclib (PD-0332991) and LIMKi3 (CAS number 1338247-35-0) were obtained from Selleck (Munich, Germany) or Merck Millipore (Burlington, Massachusetts, USA), respectively.

Jensen P., Carlet M., Schlenk R.F., Weber A., Kress J., Brunner I., Słabicki M., Grill G., Weisemann S., Cheng Y.Y., Jeremias I., Scholl C, & Fröhling S. (2020). Requirement for LIM kinases in acute myeloid leukemia. Leukemia, 34(12), 3173-3185.

+ Open protocol

+ Expand

Lentiviral Expression Analysis in K562 and HSPCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

For lentiviral expression analysis, K562 cells were plated in 24-well plates at 5 × 10⁴ cells/well and treated with a range of vector doses to obtain populations with 10% transduction or lower, thus ensuring that the majority of cells received only single integrations. Cells were cultured for 1–2 weeks before flow cytometric analysis to dilute out non-integrated vector and to allow fluorescent protein levels to reach steady state.
For expression analysis in primary human CD34+ HSPCs from mobilized peripheral blood, cryopreserved cells were thawed and prestimulated overnight in X-VIVO 15 medium (Lonza) supplemented with 50 ng/ml human fms-like tyrosine kinase 3 (FLT-3) ligand, 50 ng/ml human stem cell factor and 50 ng/ml human thrombopoietin (PeproTech, Rocky Hill, NJ, USA). Viral vector was then added in an equal volume of the same medium to achieve a final vector concentration of 3 × 10⁵ transducing units/ml, as determined by transduction of K562 cells. This vector dose yielded ∼10% transduction. Twenty-four hours after vector addition, 2 ml of myeloid differentiation medium was added, composed of IMDM supplemented with 20% foetal bovine serum, 0.5% bovine serum albumin, 5 ng/ml human interleukin-3, 10 ng/ml human interleukin-6 and 25 ng/ml human stem cell factor (PeproTech).

Cooper A.R., Lill G.R., Gschweng E.H, & Kohn D.B. (2014). Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter. Nucleic Acids Research, 43(1), 682-690.

+ Open protocol

+ Expand

Retroviral Transduction of Purified HSCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

HSCs were purified with the Mouse Lineage Cell Depletion kit (Miltenyi Biotec) from bone marrow of 8–12-wk-old mice and cultured for 48 h in DMEM supplemented with 15% heat-inactivated FCS (HyClone), 1 mM Na-pyruvate, 10 ng/ml mouse IL-3 (mIL-3), 10 ng/ml mIL-6, 100 ng/ml mouse stem cell factor, 100 ng/ml mFlt3L, and 50 ng/ml human thrombopoietin (all cytokines are from PeproTech). 6-well plates (BD) were coated overnight at 4°C with 200 µl/cm² of a 50-µg/ml RetroNectin solution (Takara Bio Inc.) in PBS before retroviral loading for 4 h at 37°C in the presence of 4 µg/ml polybrene (H9268; Sigma-Aldrich). Lin⁻ cells were incubated for 4 h at 37°C in virus-bound wells at 10⁶ cells/ml, detached by manual rubbing, washed twice, resuspended in PBS, and injected (1–2 × 10⁶ cells in 200 µl) through the retroorbital sinus in 8–20-wk-old Rag2(B6)-deficient mice irradiated 8 h before transfer (4.5 Gy; RS-2000 X-Ray; RadSource Technologies).

Girelli Zubani G., Zivojnovic M., De Smet A., Albagli-Curiel O., Huetz F., Weill J.C., Reynaud C.A, & Storck S. (2017). Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs. The Journal of Experimental Medicine, 214(4), 1169-1180.

+ Open protocol

+ Expand

Transduction of Mouse Hematopoietic Stem Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

c-kit⁺ or lineage negative (Lin^–) cells isolated from BM of transgenic mice were enriched by using CD117 or Lin^– microbeads and magnetic-activated cell sorting separation columns (all from Miltenyi Biotec) according to the manufacturer’s protocol. After enrichment, cells were pre-stimulated for 24 h in StemSpan serum-free expansion medium (Stem Cell Technologies), supplemented with penicillin/streptomycin (Gibco), murine stem cell factor (100 ng/mL; PeproTech), and human thrombopoietin (50 ng/mL; PeproTech) in six-well plates at the concentration of 0.5x10⁶ cells/mL. For transduction, retronectin-coated (20 ng/mL; Takara) 12-well plates were preloaded with the viral vectors (multiplicity of infection [MOI]=5-10), followed by seeding of 0.5x10⁶ cells into each well filled with 1 mL pre-stimulation medium.

Liu Y., Dahl M., Debnath S., Rothe M., Smith E.M., Grahn T.H., Warsi S., Chen J., Flygare J., Schambach A, & Karlsson S. (2021). Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector. Haematologica, 107(2), 446-456.

+ Open protocol

+ Expand

Isolation and Transduction of CD34+ HSPCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

All BM aspirates were obtained from voluntary healthy donors supplied by AllCells (Alameda, CA, USA). BM mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. CD34⁺ HSPCs were enriched using a CD34⁺ MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched CD34⁺ HSPCs were cryopreserved in fetal bovine serum supplemented with 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) in liquid nitrogen. Cells were thawed and plated on non-tissue culture-treated six-well plates pre-coated with RectroNectin (20 μg/mL, Takara Shuzo, Otsu, Japan) at 1 × 10⁶ cells/mL. Cells were pre-stimulated for 16–24 hours in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 1× glutamine, penicillin, and streptomycin (Gemini Bio-Products, Sacramento, CA, USA), human SCF (50 ng/mL), human Flt-3 ligand (50 ng/mL), human thrombopoietin (50 ng/mL), and human interleukin-3 (20 ng/mL; all cytokines were acquired from PeproTech, Rocky Hill, NJ, USA). Concentrated viral supernatants were used at various MOIs to transduce CD34⁺ HSPCs for 24 h. These cells were washed, re-plated, and cultured under myeloid or erythroid culture conditions, as described by Romero et al.⁶⁶ On day 14 of culture, gDNA and/or mRNA was extracted from transduced cells.

Morgan R.A., Ma F., Unti M.J., Brown D., Ayoub P.G., Tam C., Lathrop L., Aleshe B., Kurita R., Nakamura Y., Senadheera S., Wong R.L., Hollis R.P., Pellegrini M, & Kohn D.B. (2020). Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences. Molecular Therapy. Methods & Clinical Development, 17, 999-1013.

+ Open protocol

+ Expand

Quantifying Hematopoietic Progenitor Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Colony-formation assays were performed as follows. For CFU-Mk measurement, 100.000 unfractionated BM cells from young and old Vwf-EGFP mice were plated per slide in quadruplicate in Mega-Cult-C media (Stem Cell Technologies, Vancouver, Canada) supplemented with 50 ng ml⁻¹ human thrombopoietin, 10 ng ml⁻¹ murine interleukin-3, 20 ng ml⁻¹ human interleukin 6 and 50 ng ml⁻¹ human interleukin 11 (Peprotech). Cultures were acetone-fixed and acethylcolinesterase (AchE) stain was performed as per manufacturer's instructions. AchE⁺ colonies were scored at day 11. For CFU-GM measurement, 50.000 unfractionated BM cells from young and old Vwf-EGFP mice were plated in M3534 media in triplicate and colonies scored at day 8. For CFU-B measurement, 200.000 unfractionated BM cells from young and old Vwf-EGFP mice were plated in triplicate in M3630 media and colonies scored at day 8. All media for CFU assays were from Stem Cell Technologies (Vancouver, Canada).

Grover A., Sanjuan-Pla A., Thongjuea S., Carrelha J., Giustacchini A., Gambardella A., Macaulay I., Mancini E., Luis T.C., Mead A., Jacobsen S.E, & Nerlov C. (2016). Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells. Nature Communications, 7, 11075.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!