Peripheral blood mononuclear cells were prepared and cultured in serum-free X-VIVO 15 media (Life Technologies, Grand Island, New York) with UFH, branded enoxaparin (Lot number EEA1412E), and the US-generic enoxaparins produced by Sandoz (Lot number 917499) and Amphastar (Lot number EP002B2) at a dose of 10 µg/mL for 1 or 5 days. Platelet factor 4 alone (5 and 10 µg/mL) was used as the negative control. As a positive control, 50 ng/mL of the toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS; Escherichia coli 0111: B4 lipopolysaccharide, Sigma-Aldrich, St Louis, Missouri), and 10 µg/mL of the TLR7/8 agonist, R848 (Invivogen, San Diego, California), were added to the constructs. The cells were harvested, washed, and labeled for viability with LIVE/DEAD Aqua (Invitrogen, Eugene, Oregon). The cells were then labeled with a multicolor antibody panel specific for cluster of differentiation (CD) 14, human leukocyte antigen-DR, antigen-presenting cell (APC) activation/maturation markers (CD86 and CD83), and lymphocyte markers (CD3 and CD19). All antibodies were purchased from eBiosciences (San Diego, California) or BD/Biosciences (San Jose, California). Data were acquired on a BD FORTESSA II flow cytometer (BD/Biosciences) and analyzed using FlowJo software (TreeStar Inc, Ashland, Oregon).
Xvivo 15 media
Manufactured by Thermo Fisher Scientific
The Xvivo 15 media is a cell culture medium designed for the maintenance and growth of various cell types in a controlled laboratory environment. It provides the necessary nutrients and growth factors to support cell viability and proliferation.
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Lab products found in correlation
Human ab serum, by Valley Biomedical (4 mentions)
Zombie yellow dye, by BioLegend (3 mentions)
Cell proliferation dye efluor 670, by Thermo Fisher Scientific (3 mentions)
Na ve pan t cell isolation kit, by Miltenyi Biotec (3 mentions)
Ab serum, by Thermo Fisher Scientific (2 mentions)
Cd3 apc cy7, by Cytek Biosciences (2 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Fortessa 2 flow cytometer, by BD (1 mentions)
Lipopolysaccharide escherichia coli 0111 b4, by Merck Group (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
X vivo 15 media, by Lonza (1 mentions)
Human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Cts immune cell sr, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Human ab serum, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Golgiplug, by BD (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Round bottom ultra low attachment 96 well plate, by Corning (1 mentions)
10 protocols using xvivo 15 media
1
Immune Response to Heparin Therapies
2
Cryopreserved PBMC CD22-CAR T-cell Generation
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Cryopreserved human PBMCs (ALLCELLS) were thawed and plated at a density of 1 × 106 cells/ml in X-vivo-15 media (Lonza) supplemented with 5% human AB serum (Gemini) or CTS Immune Cell SR (Thermo Fisher Scientific) and 20 ng/ml IL-2 (Miltenyi Biotech) for an overnight culture at 37°C. The next day, the PBMCs were activated using human T activator CD3/CD28 (Life Technology) in serum-free X-vivo-15 media without IL-2. One million activated PBMCs (in 600 μl) were immediately incubated without removing the beads in an untreated 12-well plate pre-coated with 30 μg/ml Retronectin (Takara) in the presence of lentiviral particles encoding the CD22 targeting CAR for 2 h at 37°C. Six hundred microliters of 2× X-vivo-15 media (X-vivo-15, 10% human AB serum and 40 ng/ml IL-2) was added after 2 to 3 h, and the cells were incubated at 37°C for 72 h.
3
PBMC Expansion with Peptide Stimulation
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PBMCs were incubated overnight (37°C, 5% CO2) in X-vivo 15 media (Fisher Scientific) supplemented with 5% AB Serum (Invitrogen) and a mixture of relevant peptides at a concentration of 0.5 μM of each peptide. The cells were harvested and washed, and subsequently plated in 24-well plates at a concentration of 5 × 106/ml supplemented with 50 U/ml IL-2 for expansion. Fresh media and IL-2 were supplemented every second day until the cells were harvested at day 8, and IL-15 (15 ng/ml) was added the last 4 days.
4
PBMC Expansion via Peptide Stimulation
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PBMCs were incubated overnight (37 o C, 5% CO 2 ) in X-vivo 15 media (Fisher Scientific) supplemented with 5% AB Serum (Invitrogen) and a mixture of relevant peptides at a concentration of 0.5µM of each peptide. The cells were harvested and washed, and subsequently plated in 24-well plates at a concentration of 5 x 10 6 /ml supplemented with 50U/ml IL-2 for expansion. Fresh media and IL-2 were supplemented every second day until the cells were harvested at day 8, and IL-15 (15ng/ml) was added the last four days.
5
Profiling T cell subsets in PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient separation (Ficoll-Paque, GE Healthcare), and cryopreserved on the day of collection. Thawed PBMCs were allowed to recover in culture overnight, and then plated at 1.5 × 106 cells/ml in X-VIVO 15 media (ThermoFisher), supplemented with 5 % human AB serum (Life Technologies). Cells were stimulated with PMA (50 ng/ml) + ionomycin (0.5 μg/ml), or media alone, for 5 h in the presence of brefeldin A (GolgiPlug, BD Biosciences). Treg frequency and intracellular cytokine production by T cells were evaluated by flow cytometry. Treg (CD4+CD25hiCD127lowFoxP3+) frequency was expressed as a proportion of total CD3+ T cells. Frequencies of the following CD4+ T cell subsets were quantitated as: Th1 (IFN-γ+), Th17 (IL-17+), Th2 (IL-4+), and Tr1 (IL-10+). Total (CD3+) T cells and CD4+ T cells were reported as proportions of PBMC and total T cell populations, respectively.
6
Stimulating Primary T Cell Growth
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Primary T cells from three healthy donors were purchased from Astarte Biologics and grown in X-VIVO™ 15 media with Human T Activator CD3/CD28 Dynabeads (Thermo Fisher Scientific #11131D) at a 1:1 ratio of beads to cells. The culture media was also supplemented with IL-2 at 0.5 ng/mL to stimulate T cell growth. Beads were replaced every 7-10 days during passaging, while IL-2 was added every 2-3 days. Transfections of primary T cells followed the protocol that was optimized for the Jurkat T cells (2.75 µL Lipofectamine and 1 µg of pDNA per well in a 24-well plate).
7
Dendritic Cell Isolation and Allogeneic Stimulation
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Mixed cell suspensions were centrifuged by standard Ficoll gradient as described previously [9 , 17 (link), 27 ], prior to DC isolation using positive magnetic bead selection with either the CD14 + or CD1a + isolation kits (Miltenyi Biotec) according to the manufacturer’s instructions. After two rounds of positive selection, purity of the CD14 + and CD1a + population was about 90% [9 , 17 (link), 27 ]. Isolated DCs were plated in round bottom ultra-low attachment 96-well plates (Corning, Corning, NY) in Xvivo15 media (Invitrogen Waltham, MA) supplemented with 10% human AB serum (Valley Biomedical Winchester, VA) for in vitro allogeneic stimulation.
8
Naïve T Cell Proliferation Assay with Mucosal APCs
9
Naïve T Cell Proliferation Assay with Mucosal APCs
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Blood naïve T cells were purified after ficoll gradient using the Naïve Pan T Cell Isolation Kit (Miltenyi Biotec) and stained with Cell Proliferation Dye eFluor-670 (eBioscience) as recommended by the manufacturer. Purified mucosal CD1a+ or CD14+ were plated with naïve T cells (1:15) in round-bottom 96-well plates, in Xvivo 15 media (Invitrogen) supplemented with 10% human AB serum (Valley Biomedical). After 6 days in culture, proliferation of T cells was assessed by flow cytometry after staining with zombie yellow dye (Biolegend) and CD3-APC-Cy7 (Tonbo). As a control, blood monocyte-derived DC were generated in vitro with IL-4 and GM-CSF66 (link) after CD14+ selection as previously described16 (link).
10
Naive T Cell Proliferation Assay
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Naïve T cells were purified from cryopreserved peripheral blood mononuclear cells (PBMCs) using the Naïve Pan T Cell Isolation Kit (Miltenyi Biotec). After purification, isolated naïve T cells were >99% CCR7+ CD45A+ as determined by flow cytometry with CD3‐APC‐Cy7 (Tonbo), CCR7‐PE‐Cy7 (Miltenyi Biotec), and CD45RA‐APC (Biolegend). Naïve T cells were stained with Cell Proliferation Dye eFluor‐670 (eBioscience) as recommended by the manufacturer. Purified mucosal CD1a+ or CD14+ cells (5 × 103 cells) were plated with naïve T cells (7.5 × 104 cells) (1:15 ratio) in round‐bottom 96‐well plates, in Xvivo 15 media (Invitrogen) supplemented with 10% human AB serum (Valley Biomedical). After 6 days in culture, proliferation of T cells was assessed by flow cytometry after staining with zombie yellow dye (Biolegend) and CD3‐APC‐Cy7, CD8‐FITC (Tonbo), CD4‐PE, CD103‐PE‐Cy7 (eBiosciences), and CD11c‐PerCp‐Cy5.5 (Biolegend). Naïve T cells alone were used as a negative control. For some experiments, TGFβ receptor 1 blocker, SB431542 (10 μmol/L, Tocris Cookson Inc) (Ochiel, Ochsenbauer, et al., 2010 ), was added to the cultures at the beginning of each experiment.
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