X vivo 15 medium

Manufactured by Lonza

Sourced in Switzerland, United States, Belgium, Germany, China, United Kingdom, France, Sweden, Canada

X-VIVO 15 medium is a serum-free, protein-free cell culture medium designed for the growth and maintenance of a wide variety of cell types, including human and animal cells. It is formulated to support the proliferation and differentiation of cells without the need for serum or other animal-derived components.

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Lab products found in correlation

408 protocols using x vivo 15 medium

Assessing Tumor-Specific T Cell Cytotoxicity

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In brief, TCC88 (2 × 10⁴ cells per well) was stimulated using the tumour antigen SIN3A* and Victivallis gut bacterium HGM3 peptides (2 µM) separately on irradiated autologous PBMCs (1 × 10⁵ cells per well) for 72 h in X-VIVO 15 medium (Lonza). Next, the cells were pooled and washed once with X-VIVO 15 medium (Lonza). Autologous glioblastoma cells were stained with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) for 3 min and immediately washed three times with serum-containing medium. Target cells were then resuspended in X-VIVO 15 medium (Lonza) and seeded at 2 × 10⁴ cells per well. Activated and rested TCC88 cells were co-cultured at 1:1 and 5:1 effector to target ratios in duplicates. Next, the plate was centrifuged at 80g for 1 min to ensure cell–cell contact. After 24 h, supernatants were removed and cells were detached using accutase (Thermo Fisher Scientific), washed and stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Scientific), and the percentage of PKH26⁺Near-IR⁺ dead tumour cells was assessed using BD LSR Fortessa. HLA-DR expression of cancer cells was also measured using PE-Cy7 anti-human HLA-DR antibody (1:50) (BioLegend). To calculate the specific T cell-mediated cytotoxicity, target cells were separately seeded without TCC88 and their death (%) was considered as unspecific background death.

Naghavian R., Faigle W., Oldrati P., Wang J., Toussaint N.C., Qiu Y., Medici G., Wacker M., Freudenmann L.K., Bonté P.E., Weller M., Regli L., Amigorena S., Rammensee H.G., Walz J.S., Brugger S.D., Mohme M., Zhao Y., Sospedra M., Neidert M.C, & Martin R. (2023). Microbial peptides activate tumour-infiltrating lymphocytes in glioblastoma. Nature, 617(7962), 807-817.

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Ex Vivo Expansion of LSK Cells

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For 11-day LSK cultures, LSK cells (Lin⁻c-Kit⁻ Sca-1⁺) were sorted to purity and cultured in 96-well plates with 4000 cells per well in X-VIVO 15 medium (Lonza) with 2 ng/mL thrombopoietin (TPO) and stem cell factor (SCF) and 5 ng/mL Flt3-L. LSK cultures contained 25% supernatant from 16 h co-cultures of BM DCs with and without zymosan in X-VIVO 15 medium. In some experiments, 5 µg/mL G-CSF blocking antibody (clone 67604; R&D Systems, Minneapolis, MN, USA) or Rat IgG1 isotype control (Biolegend, San Diego, CA, USA) was added. Cells were cultured at 37 °C in a humidified incubator at 5% CO₂. All cytokines were obtained from Peprotech (London, UK).

Goedhart M., Slot E., Pascutti M.F., Geerman S., Rademakers T., Nota B., Huveneers S., van Buul J.D., MacNamara K.C., Voermans C, & Nolte M.A. (2021). Bone Marrow Harbors a Unique Population of Dendritic Cells with the Potential to Boost Neutrophil Formation upon Exposure to Fungal Antigen. Cells, 11(1), 55.

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Retaining CD8+ T Cells in Glucose/Galactose Media

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Example 5

The retention of CD8+ T cell subsets in glucose/galactose serum-free medium was analyzed. Primary human T cells from three normal donors were isolated from PBMCs with DYNABEADS® UNTOUCHED™ Human T cells. T cells were seeded at a density of 1×10⁶/mL and were activated with DYNABEADS® Human T-Expander CD3/CD28 at a ratio of 3 beads per T cell. Cells were cultured in Lonza X-VIVO™ 15 medium alone, Lonza X-VIVO™ 15 medium with 5% human AB serum, serum-free medium containing a 1:1 mixture of glucose and galactose alone or supplemented with 5% human AB serum. T cells were counted on a Beckman-Coulter ViCell analyzer and fed to a cell density of 5×10⁵on day 3, 5, 7, 10 and 12. T cells were stained with antibodies against CD3, CD4 and CD8 prior to stimulation and after 10 days post-expansion. Frequency of CD8+ T cells post expansion was compared to the frequency before expansion and expressed as a percentage. Results compiled from multiple independent experiments with cells derived from 9 normal donors. As depicted in FIG. 9, results show 99% retention of CD8+ T cells post expansion in glucose/galactose serum-free medium when compared to the frequencies in control media. Serum supplementation has a slight negative effect in glucose/galactose medium when compared to control media which shows similar CD8+ T cell frequencies irrespective of serum supplementation.

US11597911B2. Expansion of populations of T cells by the use of modified serum free media (2023-03-07). Life Technologies Corporation [US], The Trustees of the University of Pennsylvania [US]. Inventors: Angel M. Varela-Rohena [US], Melanie B. Andolina [US], James L. Riley [US], Andrew Medvec [US].

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Cytotoxicity Assay of Effector Cells

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Effector TILs or TCCs were stimulated with pooled peptides for 5 days or 4 days, respectively, using irradiated autologous PBMCs as APCs. TILs and TCCs were then harvested, washed, and resuspended with X-VIVO 15 medium (Lonza). Tumor target cells were detached with accutase (Thermo Fisher Scientific), washed, and labeled with a PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) for 3 minutes. After labeling, cells were washed three times with serum-containing medium, resuspended with X-VIVO 15 medium (Lonza), adjusted to 2 × 10⁵ cells/mL, and added in 100 μL/well in 96-well U-bottom plates (Greiner Bio-One). Effector TILs or TCCs were added to respective wells containing PKH26-labeled target tumor cells at 1:1, 2:1, 5:1, and 10:1 effector-to-target ratios. Plates were centrifuged at 80 × g for 1 minute to improve effector-target cell contact and incubated for 24 hours. After incubation, plates were centrifuged at 250 × g for 5 minutes, supernatants removed, and cells detached with accutase (Thermo Fisher Scientific). Cells were harvested, stained with a LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific), and analyzed using a BD LSR Fortessa to analyze PKH26⁺Near-IR⁺ dead target cells.

Wang J., Weiss T., Neidert M.C., Toussaint N.C., Naghavian R., Sellés Moreno C., Foege M., Tomas Ojer P., Medici G., Jelcic I., Schulz D., Rushing E., Dettwiler S., Schrörs B., Shin J.H., McKay R., Wu C.J., Lutterotti A., Sospedra M., Moch H., Greiner E.F., Bodenmiller B., Regli L., Weller M., Roth P, & Martin R. (2022). Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma. Clinical Cancer Research, 28(24), 5368-5382.

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Macrophage Differentiation and Modulation

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To obtain M1 macrophages, freshly isolated monocytes were incubated for 6 days in X-VIVO 15 medium (Lonza, Guangzhou, China) with GM-CSF(100 ng/ml, Abmole Bioscience, Houston, USA). On Day 6, LPS (10 ng/ml, Sigma-Aldrich, Shanghai, China) and IFN-γ (50 ng/ml, Sino Biological, Beijing, China) were added, and the cells were then incubated for an additional 2 days. To obtain M2 macrophages, freshly isolated monocytes were incubated for 6 days in X-VIVO 15 medium (Lonza, Guangzhou, China) with M-CSF (100 ng/ml, Sino Biological). On Day 6, IL-4, IL-10 and TGF-β (20 ng/ml, Sino Biological) were added, and the cells were further incubated for 2 days. In some experiments, the Rac1 inhibitor NSC23766 (30 µM, Abmole Bioscience) was added to the freshly isolated monocytes and incubated for 8 days with cells. In some experiments, the TLR4 signaling inhibitor resatorvid (40 nM, Abmole Bioscience) was added to the cell culture on Day 6.

Fu H., Zhang P., Zhao X.D, & Zhong X.Y. (2023). Interfering with Rac1-activation during neonatal monocyte-macrophage differentiation influences the inflammatory responses of M1 macrophages. Cell Death & Disease, 14(9), 619.

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Activation and Enrichment of Recipient T Cells

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Buffy coats were donated from healthy volunteers. PBMCs were isolated by density gradient centrifugation. T cells were purified using the human T cell enrichment cocktail (STEMCELL RosetteSep), activated with Dynabeads (1:1 beads:cell) Human T-Activator CD3/CD28 (ThermoFisher), and cultured in X-vivo 15 medium (Lonza) supplemented with 10% FBS, 5ng/ml recombinant human interleukin (IL)-7 and IL-15 (BioLegend). The medium was changed to retain the cells at a density of 0.25-1 × 10⁶ cells/ml. To obtain activated RTCs, recipient PBMCs were firstly stimulated with irradiated PBMCs from anther donor at a ratio 1:1 for 6 to 7 days in X-vivo 15 medium (Lonza) supplemented with 10% FBS and 20 U/ml IL-2 (Pepro Tech). RTCs were subsequently enriched from the stimulated recipient PBMCs using MojoSort™ Human CD3 T Cell Isolation kit (BioLegend).

Zhang Y., Fang H., Wang G., Yuan G., Dong R., Luo J., Lyu Y., Wang Y., Li P., Zhou C., Yin W., Xiao H., Sun J, & Zeng X. (2023). Cyclosporine A-resistant CAR-T cells mediate antitumour immunity in the presence of allogeneic cells. Nature Communications, 14, 8491.

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Primary Thymic Epithelial Cell Culture and Manipulation

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Primary thymic epithelial cells (TECs) were cultured from thymic explants and correspond mainly to medullary TECs as previously described [21] . TECs were transfected with Lipofectamine RNAiMax (Life Technologies). Briefly, 40pmol of miR-7-5p or scramble RNA (Eurogentec, Seraing, Belgium) were diluted in 50µl of X-vivo15 medium (Lonza, Switzerland).
In parallel, 4µl of Lipofectamine RNAiMax were diluted in 50µl of X-vivo15 medium. Then, both solutions were mixed and incubated for 20 minutes at room temperature. The resulting mixes were added to cultured cells (4 x10 5 cells per well) at a ratio of 1:4 of X-vivo15 medium in 12well plates for 48h.
For methylation experiments, TECs were seeded in RPMI containing 10% of fetal calf serum (5 x10 5 cells per well) and treated after 24 hours with 5µM of 5-Azacytidine for 48 hours. TECs were then lysed for total RNA extraction.

Cron M.A., Maillard S., Delisle F., Samson N., Truffault F., Foti M., Fadel E., Guihaire J., Berrih-Aknin S, & Le Panse R. (2018). Analysis of microRNA expression in the thymus of Myasthenia Gravis patients opens new research avenues. Autoimmunity reviews, 17(6).

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Neutrophil Functional Assays Optimization

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Human and mouse neutrophils were seeded on 12-mm glass cover slips in X-VIVO^TM 15 medium (Lonza, Walkersville, MD, USA), primed with 25 ng per ml GM-CSF for 20 min, and subsequently stimulated with 10⁻⁸ M C5a, 100 ng per ml LPS or co-cultured with Escherichia (E.) coli GFP M655 (E. coli-GFP; a kind gift of E. Slack, ETH Zurich) for 15 min. In selected experiments, the following inhibitors were used 30 min before GM-CSF priming: Oligomycin A (2.5, 5, or 10 µg per ml), 2-DG (1, 3, 5 mM), rotenone (10 µM), antimycin A (5 µg per ml), Q-VD (20 µM), taxol (1 µM) and nocodazole (5 µM). In other experiments, cells were pre-cultured for 30 min in the presence of ATP (10, 100 µM, or 1 mM) or NMN (500 μM) and then stimulated for 45 min, besides GM-CSF/C5a, also with RNP-ICs-SLE [IgG, purified from with SLE antibody positive human plasma (Cat # DA1805, Trina Bioreactive AG, Nänikon, Switzerland, dilution 1:33), mixed with small nuclear ribonucleoprotein (SmRNP) (Cat # ATR01-02, Arotec, New Zealand, dilution 1:100)] or RNP-ICs-Ab [an anti-damaged DNA/RNA monoclonal antibody (clone 15A3, Cat # SMC-155, StressMarq Biosciences, Victoria, Canada, dilution 1:33) mixed with SmRNP]^{29 (link)} or P. aeruginosa (ATCC-BAA-47; strain HER-1018)^{42 (link)}; with a multiplicity of infection of 10:1 (bacteria to neutrophils).

Amini P., Stojkov D., Felser A., Jackson C.B., Courage C., Schaller A., Gelman L., Soriano M.E., Nuoffer J.M., Scorrano L., Benarafa C., Yousefi S, & Simon H.U. (2018). Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production. Nature Communications, 9, 2958.

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Th Cell Differentiation with OX40L

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Naïve CD4 T cells were cultured for 5 days in X-VIVO^TM 15 medium (Lonza) with only anti-CD3/anti-CD28 Dynabeads (Life Technologies) to obtain Th0, or in combination with either 10 ng/mL IL-12 (R&D Systems) to obtain Th1, 25 ng/mL IL-4 (R&D Systems) to obtain Th2, or a cocktail of 100 ng/mL IL-23 (R&D Systems), 10 ng/mL IL-1β, 1 ng/mL TGF-β and 20 ng/mL IL-6 (Peprotech) to obtain Th17 as already published^{6 (link)}. When indicated, 600 ng/mL rhOX40L (R&D Systems) was added to the T cell culture. At the end of the culture T cells were washed, counted and reseeded at 10⁶ cells/mL and restimulated with anti-CD3/CD28 Dynabeads (Life Technologies) for 24 h before collecting supernatants for cytokine measurement.

Karpf L., Trichot C., Faucheux L., Legbre I., Grandclaudon M., Lahoute C., Mattoo H., Pasquier B, & Soumelis V. (2022). A multivariate modeling framework to quantify immune checkpoint context-dependent stimulation on T cells. Cell Discovery, 8, 1.

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Primary Human CD14+ Monocyte Isolation and M1 Polarization

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Primary human CD14⁺ monocytes were isolated from buffy coats (3 female donors, age: 38 ± 12) by density gradient separation using Ficoll Paque Plus (GE Healthcare Life Sciences, Piscataway, NJ, USA) followed by magnetically activated cell sorting using human anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), as described previously [28 (link)]. The isolated CD14⁺ monocytes were seeded into a 96-well plate at a density of 500,000 cells/cm² in X-VIVO^TM 15 medium (Lonza, Basel, Switzerland) with 10% heat-inactivated FBS and 100 U/mL penicillin and streptomycin. After cell attachment (approx. 1 h after seeding), cells were stimulated for 72 h (renewed after 48 h) with 10 ng/mL recombinant human TNF (PeproTech, Cranbury, NJ, USA) and 10 ng/mL recombinant human IFNγ (PeproTech) for polarization towards an M1-like phenotype of cells.

Muenzebrock K.A., Ho F.Y., Pontes A.P., Jorquera-Cordero C., Utomo L., Garcia J.P., Willems P.C., Welting T.J., Rip J, & Creemers L.B. (2024). Polymeric Nanoparticles Enable mRNA Transfection and Its Translation in Intervertebral Disc and Human Joint Cells, Except for M1 Macrophages. Pharmaceutics, 16(4), 438.

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