Cc100 cytokine cocktail

Manufactured by STEMCELL

Sourced in Canada

The CC100 cytokine cocktail is a combination of growth factors and cytokines formulated to support the culture and maintenance of human induced pluripotent stem cells (hiPSCs). It is designed to promote the self-renewal and pluripotency of hiPSCs in feeder-free culture conditions.

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CC100 (16 citations) StemSpan CC100 cytokine cocktail (10 citations) Stem Span Cytokine Cocktail CC100 (2 citations)

7 protocols using cc100 cytokine cocktail

Colony-Forming Assay for Human HSCs

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For human CD34⁺ HSC CFU assays, freshly purified cells were seeded at 5,000 cells/well in human methylcellulose medium (methocult H4230; STEMCELL Technologies) supplemented with CC100 Cytokine Cocktail. Cells were plated in 35-mm petri dishes and cultured in a fully humidified atmosphere with 5% CO₂ at 37°C for 14 days. The number of colonies was scored after 14 days. For FACS analysis of the CFUs, colonies were harvested, washed twice with PBS, and evaluated for EGFP expression.

El Ashkar S., Van Looveren D., Schenk F., Vranckx L.S., Demeulemeester J., De Rijck J., Debyser Z., Modlich U, & Gijsbers R. (2017). Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy. Molecular Therapy. Nucleic Acids, 7, 231-245.

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Culturing U937 and CD34+ Cells

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The U937 cell line (ATCC, Manassas, VA, USA) was cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies). Primary CD34⁺ cells were obtained from human umbilical cord blood collected at Skåne University Hospital. The mononuclear cell population was isolated by separation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) and CD34⁺ cells were subsequently selected using the Indirect CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were grown in StemSpan SFEM medium (Stemcell Technologies, Vancouver, Canada) supplemented by 20% fetal bovine serum and the CC100 cytokine cocktail (Stemcell Technologies).

Sandén C., Järvstråt L., Lennartsson A., Brattås P.L., Nilsson B, & Gullberg U. (2014). The DEK oncoprotein binds to highly and ubiquitously expressed genes with a dual role in their transcriptional regulation. Molecular Cancer, 13, 215.

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Lentiviral Expression of ANKRD26 Variants

Cited 1 time

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cDNA encoding full-length or truncated exon11+ ANKRD26 and a WAC-ANKRD26 fusion starting from exon 10 of ANKRD26 were cloned into the HMD lentiviral backbone. For production of lentiviruses, the appropriate viral packaging and genomic vectors were introduced into HEK 293T cells with calcium phosphate transfection. Viral supernatants were collected 48 h after transfection. To examine the activity of the fusion transcript-encoded cDNAs, overexpression in HSPCs was performed by transduction of relevant constructs at a multiplicity of infection of 50. HSPCs were cultured for 5 d after transduction in serum-free StemSpan II medium (STEMCELL Technologies) supplemented with CC100 cytokine cocktail (STEMCELL Technologies), 50 ng/ml TPO (PeproTech), and 35 nM UM171 (Bao et al., 2020 (link)). To assess for effects upon signaling activity downstream of the TPO receptor, 96 h after transduction cells were serum starved for 2 h followed by stimulation with TPO at 50 ng/ml for 15 min and subjected to downstream analysis.

Wahlster L., Verboon J.M., Ludwig L.S., Black S.C., Luo W., Garg K., Voit R.A., Collins R.L., Garimella K., Costello M., Chao K.R., Goodrich J.K., DiTroia S.P., O’Donnell-Luria A., Talkowski M.E., Michelson A.D., Cantor A.B, & Sankaran V.G. (2021). Familial thrombocytopenia due to a complex structural variant resulting in a WAC-ANKRD26 fusion transcript. The Journal of Experimental Medicine, 218(6), e20210444.

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Isolation and Expansion of Primary CD4+ T Cells and CD34+ HSCs

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Human peripheral blood mononuclear cells (PBMCs) were purified from buffy coats of three different donors, obtained from the Red Cross blood transfusion center, using density-gradient centrifugation (Lymphoprep; Axis-Shield). Primary CD4⁺ T cells were selectively enriched using bi-specific monoclonal antibody (mAb) CD3.8 (0.5 μg/mL, NIH AIDS Reagents Program; https://www.aidsreagent.org) for 5 days. CD4⁺ T cells were cultured in RPMI medium supplemented with 15% FBS, gentamycin, interleukin (IL)-2 (100 U/mL; Peprotech), and MEM Non-Essential Amino Acids Solution (MEM NEAA) (50 μg/mL; Gibco-BRL), referred to as T-cell medium (TCM). CD34⁺ HSCs were positively selected with anti-CD34-conjugated microbeads according to the manufacturer’s instructions (MACS; Miltenyi Biotec) and stimulated for 48 hr in StemSpan SFEMII medium containing CC100 Cytokine Cocktail (STEMCELL Technologies).

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Clonogenic Assay for Leukemic Cells

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Kasumi-1, KG-1, ME-1, HEL, U937, SKM-1, Nomo-1, MOLM-13, OCI-AML3 and K562 cells were grown in RPMI-1640 medium supplemented with FBS (10–20% final concentration, Sigma-Aldrich Ltd). Human CD34+ cells were maintained in liquid culture using StemSpan Serum-free media and CC100 cytokine cocktail (STEMCELL Technologies, Vancouver, BC, Canada). All growth media also contained 1% penicillin/streptomycin. Unless otherwise stated, mouse BM, MT2 and NHA9 mouse immortalized progenitors, leukemic cell lines and human control CD34⁺ and AML patient primary cells were plated at a concentration of 10 000–20 000 cells/plate (in duplicate) using the MethoCult H4531, MethoCult H4435 Enriched (for human cells) or MethoCult GF M3434 (for mouse cells) (STEMCELL Technologies). Colonies were scored at 7–12 days.

Giotopoulos G., Chan W.I., Horton S., Ruau D., Gallipoli P., Fowler A., Crawley C., Papaemmanuil E., Campbell P., Göttgens B., Van Deursen J., Cole P, & Huntly B. (2015). The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia. Oncogene, 35(3), 279-289.

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CD34+ Cell Isolation and Differentiation

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Bone marrow (BM)-derived CD34+ were obtained from Calabretta’s lab at Thomas Jefferson University. CD34+ CD33− cells were sorted at the Flow Cytometry facility at Wistar Institute. Fetal liver (FL)-derived CD34+ cells were obtained from Stem Cell and Xenograft Core at University of Pennsylvania and were maintained in StemSpan SFEM medium supplemented with CC100 cytokine cocktail (Stem Cell Technologies). De-identified human cord blood (CB) was obtained from volunteers with informed consent at Helen F. Graham Cancer Center and Research Institute at Christiana Hospital. Mononucleated cells (MNC) were separated with Ficoll-Hystopaque Plus (GE Healthcare). CD34+ cells were then isolated using human CD34 MicroBeads Kit (Miltenyi Biotec) following manufacturer’s instructions. CD34+ were maintained in StemSpan SFEM medium supplemented with 1X CC100 cytokine cocktail. BM- and FL-derived CD34+ were used for CFU assay, BM-, FL- and CB-derived CD34+ were in vitro differentiated and used for RNA-seq and ChIP-seq experiments, respectively. Circulating monocytes were obtained from the Human Immunology Core at University of Pennsylvania.

Barbieri E., Trizzino M., Welsh S.A., Owens T.A., Calabretta B., Carroll M., Sarma K, & Gardini A. (2018). Targeted enhancer activation by a subunit of the Integrator complex. Molecular cell, 71(1), 103-116.e7.

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Erythroid Differentiation of Umbilical Cord Blood CD34+ Cells

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Fresh umbilical cord blood (collected within 24–48 hours) was obtained from the St. Louis Cord Blood Bank (SLCBB, St. Louis, MO). GE Ficoll Paque™ Premium (GE Healthcare) was used to isolate mononuclear cells following the manufacturer’s protocol. CD34+ cells were enriched from the mononuclear cells by magnetic separation using the EasySep™ human CD34 positive selection kit (Stem Cell Technologies). CD34+ cells were expanded for 8 days in StemSpan SFEM medium (Stem Cell Technologies) containing 1X CC100 cytokine cocktail (Stem Cell Technologies), 8 μl/ml LDL (Sigma, L7914) and 2% Penicillin/Streptomycin (P/S, Gibco) [22 (link)]. The lentivirus-containing media were used to transduce CD34+ cells on expansion day 8. On the third day after infection, successfully transduced GFP+ cells were collected by FACS and differentiated along the erythroid lineage [22 (link),23 (link)]. UCB erythroblast pellets were red when harvested on differentiation day 8 (DD8).

Vinjamur D.S., Alhashem Y.N., Mohamad S.F., Amin P., Williams DC J.r, & Lloyd J.A. (2016). Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts. PLoS ONE, 11(2), e0146802.

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