X vivo 15 serum free hematopoietic cell medium

Manufactured by Lonza

Sourced in Switzerland

X-VIVO 15 is a serum-free medium designed for the culture and expansion of hematopoietic cells. It is a liquid medium formulation that provides a chemically-defined, protein-free environment for cell culture.

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16 protocols using x vivo 15 serum free hematopoietic cell medium

Expansion of NY-ESO-1 CD8+ T Cells

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Human NY-ESO-1 CD8⁺ T cells were stimulated with plate-bound anti-human CD3 (TONBO, 40-0038-U100, clone UCHT1) at 10 μg ml⁻¹ and anti-human CD28 (TONBO, 40-0289-U100, clone CD28.2) at 2 μg ml⁻¹ for 48 h before expanded with recombinant human IL-2 (BioLegend, 589106) at 100 IU ml⁻¹. Cells were cultured in completed X-Vivo medium, which is X-Vivo 15 Serum-free Hematopoietic Cell Medium (Lonza, BE02-060Q) supplemented with 5% inactivated FBS (Gibco, 10082147), 50 μM 2-mercaptoethanol (Gibco, 21985023) and 10 mM N-acetyl l-cysteine (Sigma, A7250-5G) at 1 × 10⁶ cells per ml.

Zhang Y., Vu T., Palmer D.C., Kishton R.J., Gong L., Huang J., Nguyen T., Chen Z., Smith C., Livák F., Paul R., Day C.P., Wu C., Merlino G., Aldape K., Guan X.Y, & Jiang P. (2022). A T cell resilience model associated with response to immunotherapy in multiple tumor types. Nature medicine, 28(7), 1421-1431.

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Isolation and Activation of Human T Cells

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Peripheral blood was obtained from the healthy donors. Blood sampling was performed following the required ethical procedures. Lymphocytes were isolated by density gradient centrifugation following the manual of the Human Lymphocyte Separation Medium (DAKEWE). Human T cells were purified using the human CD3 T cell isolation kit (BioLegend), stimulated with CD3/CD28 T cell Activator Dynabeads (Gibco) and cultured at 10⁶/mL in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza), supplemented with 5 ng/mL human IL-7 (PeproTech) and 5 ng/mL human IL-15 (PeproTech)^{40 (link),65 (link)}, with slight modification. 48 h after T cell stimulation, T cells were transduced with lentiviral supernatants from 293 T cell line in the presence of polybrene (6 μg/mL, Yeasen) by centrifugal infection. T cells were analyzed by flow cytometry 4 days after transduction and were used for further experiments. All human subjects were informed and signed informed consent prior to inclusion in the study and all human cell isolation and related experiments were approved by the Ethics Committee for Human Studies of Second Affiliated Hospital of Zhejiang University School of Medicine (2019 NO.388).

Chen S., Chen G., Xu F., Sun B., Chen X., Hu W., Li F., Syeda M.Z., Chen H., Wu Y., Wu P., Jing R., Geng X., Zhang L., Tang L., Li W., Chen Z., Zhang C., Sun J., Chen W., Shen H, & Ying S. (2022). Treatment of allergic eosinophilic asthma through engineered IL-5-anchored chimeric antigen receptor T cells. Cell Discovery, 8, 80.

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SARS-CoV-2 Variant Spike Peptide Stimulation Assay

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Cryopreserved PBMC (5 × 10⁶/sample) were thawed in prewarmed RPMI-1640 with L-glutamine (Lonza, Basel, Switzerland) + 10% FCS. Thawed PBMCS were rested for 3-4 h at 37 °C in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza) supplemented with 5% human Ab serum. Cells were then stimulated with ~1 nmol of peptide pool corresponding to SARS-CoV-2 spike (S) of US-WA (ancestral), P.1 (Gamma), or B.1.617.2 (Delta). Peptides were 16mer peptide pools, overlapping by 10 amino acids, purchased from 21st century Biochemicals Inc. Cell suspensions were transferred to pre-coated Human IFN-γ, IL-2, Granzyme-B (Gz-B) FLUORISpot kit plates (Mabtech, Inc.) and developed after 48 h according to manufacturer instructions. Spots were imaged and counted using a Mabtech Iris Fluorispot reader (Mabtech).

Jergović M., Uhrlaub J.L., Watanabe M., Bradshaw C.M., White L.M., LaFleur B.J., Edwards T., Sprissler R., Worobey M., Bhattacharya D, & Nikolich-Žugich J. (2022). Competent immune responses to SARS-CoV-2 variants in older adults following two doses of mRNA vaccination. Nature Communications, 13, 2891.

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Preparation of Cell Culture Media

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α-Galactosylceramide (αGC, KRN7000) was purchased from Avanti Polar Lipids. Recombinant human IL-2, IL-3, IL-4, IL-7, IL-15, Flt3-Ligand, Stem Cell Factor (SCF), Thrombopoietin (TPO), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) were purchased from Peprotech. Ganciclovir (GCV) was purchased from Sigma.
X-VIVO 15 Serum-free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Corning Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including Penicillin-Streptomycine-Glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and Sodium Pyruvate, were purchased from GIBCO. Beta-Mercaptoethanol (β-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. Complete lymphocyte culture medium (denoted as C10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 mM), Sodium Pyruvate (1 mM), β-ME (50 mM), and Normocin (100 mg/ml). Medium for culturing human MM.1S tumor cell line (denoted as R10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol). Adherent cell culture medium (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).

Li Y.R., Zhou Y., Kim Y.J., Zhu Y., Ma F., Yu J., Wang Y.C., Chen X., Li Z., Zeng S., Wang X., Lee D., Ku J., Tsao T., Hardoy C., Huang J., Cheng D., Montel-Hagen A., Seet C.S., Crooks G.M., Larson S.M., Sasine J.P., Wang X., Pellegrini M., Ribas A., Kohn D.B., Witte O., Wang P, & Yang L. (2021). Development of allogeneic HSC-engineered iNKT cells for off-the-shelf cancer immunotherapy. Cell Reports Medicine, 2(11), 100449.

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Generation of Humanized BLT and BLT-iNKT Mice

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BLT (human bone marrow-liver-thymus engrafted NSG mice) and BLT-iNKT (human iNKT TCR gene-engineered BLT mice) humanized mice were generated as previously described, with some modifications (Lan et al., 2006 (link); Melkus et al., 2006 (link); Smith et al., 2016 (link)). In brief, human CD34⁺ HSCs were cultured for no more than 48 hours in X-VIVO™ 15 Serum-free Hematopoietic Cell Medium (Lonza) containing recombinant human Flt3 ligand (50 ng/mL), SCF (50 ng/mL), TPO (50 ng/mL), and IL-3 (20 μg/mL) in non-tissue culture-treated plates coated with Retronectin (20 μg/ml) (Takara). Viral transduction, when applicable, was performed at 24 hours by adding concentrated lentivectors (Lenti/iNKT, Lenti/iNKT-EGFP, or Lenti/iNKT-sr39TK) directly to the culture medium. At around 48 hours, CD34⁺ cells were collected and i.v. injected into NSG mice (~0.5–1 × 10⁶ cells per recipient) that had received 270 rads of total body irradiation. 1–2 fragments of human fetal or postnatal thymus (~1 mm³) were implanted under the kidney capsule of each recipient NSG mouse. The mice were maintained on trimethoprim/sulfmethoxazole (TMS) chow in a sterile environment for 8–12 weeks until analysis or use for further experiments. Unless otherwise indicated, data from BLT-iNKT mice produced with human fetal thymus and Lenti/iNKT-sr39TK vector-transduced PBSCs were presented.

Zhu Y., Smith D.J., Zhou Y., Li Y.R., Yu J., Lee D., Wang Y.C., Di Biase S., Wang X., Hardoy C., Ku J., Tsao T., Lin L.J., Pham A.T., Moon H., McLaughlin J., Cheng D., Hollis R.P., Campo-Fernandez B., Urbinati F., Wei L., Pang L., Rezek V., Berent-Maoz B., Macabali M.H., Gjertson D., Wang X., Galic Z., Kitchen S.G., An D.S., Hu-Lieskovan S., Kaplan-Lefko P.J., De Oliveira S.N., Seet C.S., Larson S.M., Forman S.J., Heath J.R., Zack J.A., Crooks G.M., Radu C.G., Ribas A., Kohn D.B., Witte O.N, & Yang L. (2019). Development of Hematopoietic Stem Cell-Engineered Invariant Natural Killer T Cell Therapy for Cancer. Cell stem cell, 25(4), 542-557.e9.

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PBMC Cryopreservation and Characterization

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Cryopreserved PBMC (2–5 × 10⁶/sample) were thawed in prewarmed RPMI-1640 with L-glutamine (Lonza) + 10% FCS. Thawed PBMCS were rested overnight at 37 °C in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza) supplemented with 5% human Ab serum. Cells were stained with surface antibodies in PBS (Lonza) + 2% FCS, and then stained with the live dead fixable blue dye (Thermofisher). B cell tetramers were assembled by mixing 100 μg ml⁻¹ of C-terminal AviTagged RBD or S1 (ACROBiosystems) with 100 μg ml⁻¹ of streptavidin-PE (eBiosciences) or streptavidin-BV421 (BioLegend), respectively, at a 5:1 molar ratio in which 1/10 of the final volume of streptavidin was added every 5 min. Samples were stained for 1 h at 4 °C. List of antibodies used for flow cytometric staining in Supplementary Table I. Samples were acquired using a Cytek Aurora cytometer (Cytek) and analyzed by FlowJo software (Tree Star).

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Cell Culture and Transfection Protocol

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Peripheral blood mononuclear cells (PBMCs, TPCS#PB025C) were purchased from the miles-bio of Shanghai. Normal human embryonic kidney cell line HEK293, T cell leukemia cell line NFAT-Jurkat, human cervical cancer cell lines Hela, human ovarian cancer cell lines (Skov3) were purchased from the American Type Culture Collection. T lymphocytes were maintained in T cell growth medium (TCGM): X-VIVO 15% Serum-free Hematopoietic Cell Medium (Lonza, Switzerland) supplemented with 5% FBS 2 ng mL^-1 human recombinant IL-2 (100U mL^-1, Sigma, Germany). HEK293 were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA). NFAT-Jurkat and Hela were maintained in RPMI 1640-media (Gibco, Grand Island, NY, USA). Skov3 were maintained in McCoy’s 5A medium (Gibco, Grand Island, NY, USA). GFP- and luciferase-expressing Hela (Hela-GL) and Skov3 cells (Skov3-GL) were generated by transfection of Hela and Skov-3 cells with lentiviral supernatant containing luciferase-2A-GFP. All cells were cultured in recommended medium supplemented with 10% FBS (Gibco) in a 10% CO₂ incubator.

Yang F., Zhang F., Ji F., Chen J., Li J., Chen Z., Hu Z, & Guo Z. (2023). Self-delivery of TIGIT-blocking scFv enhances CAR-T immunotherapy in solid tumors. Frontiers in Immunology, 14, 1175920.

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Isolation and Activation of Human T Cells

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Peripheral blood was obtained from healthy volunteers. Ethical permission was granted by the School of Medicine, Zhejiang University, and all of the blood samples were handled following the required ethical and safety procedures. PBMCs were isolated by Ficoll density gradient centrifugation (Dakewe). Then T lymphocytes were purified using the Pan T Cell Isolation Kit (Miltenyi Biotec) and activated with CD3/CD28 T cell Activator Dynabeads (Thermo Fisher Scientific) and cultured in X‐VIVO15 Serum‐free Hematopoietic Cell Medium (Lonza) supplemented with 10% FBS, 1% penicillin/streptomycin, 5 ng ml⁻¹ interleukin‐7 (IL7) and 5 ng ml⁻¹ interleukin‐15 (IL15) (Novoprotein). The medium was changed every 2 days, and cells were plated at 10⁶ cells ml⁻¹.

Liao C., Wang Y., Huang Y., Duan Y., Liang Y., Chen J., Jiang J., Shang K., Zhou C., Gu Y., Liu N., Zeng X., Gao X., Tang Y, & Sun J. (2023). CD38‐Specific CAR Integrated into CD38 Locus Driven by Different Promoters Causes Distinct Antitumor Activities of T and NK Cells. Advanced Science, 10(27), 2207394.

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Activation of CD8+ T Cells for Functional Assays

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CD8⁺ T cells were isolated from PBMCs by magnetic-activated cell sorting (MACS), using the REAlease CD8 Microbead Kit (Miltenyi) according to the manufacturer’s instructions. T cells were cultured in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza) on sterile, growth-enhanced treated polystyrene flat-bottom plates (TPP) at 37 °C in a humidified incubator (5% CO₂). For activation in single and co-culture, CD8⁺ T cells were cultured on 96-well plates (2 × 10⁴ cells in 100 μL per well) and stimulated with 50 ng/mL recombinant human IL-2 (Invitrogen) and anti-CD2/CD3/CD28-coated microbeads, using the T Cell Activation/Expansion Kit (Miltenyi) according to the manufacturer’s instructions. A cell-to-bead ratio of 2:1 (1 × 10⁸ beads per mL) was used. Every 2 days, fresh medium containing the same supplements was added.

Heller S., Glaeske S., Gluske K., Paul J., Böhme A., Janzer A., Roider H.G., Montebaur A., Nicke B., Lesche R., von Ahsen O., Politz O., Liu N, & Gorjánácz M. (2023). Pan-PI3K inhibition with copanlisib overcomes Treg- and M2-TAM-mediated immune suppression and promotes anti-tumor immune responses. Clinical and Experimental Medicine, 23(8), 5445-5461.

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Generation of Immune Cell Cultures

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α-Galactosylceramide (αGC, KRN7000) was purchased from Avanti Polar Lipids. Recombinant human IL-2, IL-3, IL-4, IL-7, IL-15, Flt3-Ligand, Stem Cell Factor (SCF), Thrombopoietin (TPO), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) were purchased from Peprotech. Ganciclovir (GCV) was purchased from Sigma.
X-VIVO 15 Serum-Free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Corning Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including penicillin-streptomycin-glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and sodium pyruvate, were purchased from GIBCO. beta-mercaptoethanol (β-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. Complete lymphocyte culture medium (denoted as C10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 mM), sodium pyruvate (1 mM), β-ME (50 mM), and Normocin (100 mg/mL). Medium for culturing human Raji and HL60 tumor cell lines (denoted as R10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol). Medium for culturing HEK 293T cell line (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).

Li Y.R., Zeng S., Dunn Z.S., Zhou Y., Li Z., Yu J., Wang Y.C., Ku J., Cook N., Kramer A, & Yang L. (2022). Off-the-shelf third-party HSC-engineered iNKT cells for ameliorating GvHD while preserving GvL effect in the treatment of blood cancers. iScience, 25(9), 104859.

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