Peripheral blood samples and serum were obtained from patients with metastatic melanoma under an Institutional Review Board-approved protocol.15 (link) Samples were coded with an anonymized 5-digit number and their identity was unknown to those performing the experiments. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient with 1.077 g/mL Ficoll-Paque (GE Healthcare, Chicago, Illinois, USA). For cryopreservation, PBMCs were re-suspended in human AB serum (Omega Scientific; Tarzana, California, USA) with 10% dimethyl sulfoxide (Sigma-Aldrich, St Louis, Missouri, USA), frozen at −80°C and stored in liquid nitrogen. CD3+ and CD8+ T cells were isolated using a magnetic negative selection kit (StemCell Technologies, Vancouver, British Columbia, Canada). Cells were cultured with 10% AB human serum in beta-mercaptoethanol, non-essential amino acids, HEPES, penicillin, streptomycin and gentamicin supplemented X-VIVO media (Corning, Corning, New York, USA). Serum was obtained from whole blood patient samples, centrifuged to remove cells and frozen at −80°C until time of use.
X vivo media
Manufactured by Corning
Sourced in United States, Canada
X-VIVO media is a serum-free, protein-free cell culture medium designed for the ex vivo expansion and maintenance of a variety of cell types. It is formulated to support the growth and differentiation of cells without the addition of serum or other animal-derived components.
Automatically generated - may contain errors
2 protocols using x vivo media
1
Isolating Immune Cells from Metastatic Melanoma
2
Isolation and Culture of Melanoma TILs
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PBMC were obtained by leukapheresis from metastatic melanoma patients prior to treatment. TILs were obtained from surgical biopsies of melanoma metastases by culturing tumor fragments in 6000IU/mL IL-2 for 2–3 weeks [12 (link)]. All protocols were approved by the Institutional Review Board at H. Lee Moffitt Cancer Center (IRB 106509, 107,273; Tampa, FL). Cells were obtained under a Materials Transfer Agreement. Samples were coded with anonymized 5-digit numbers and their identity was unknown to those performing the experiments. PBMCs were separated using 1.077g/mL Ficoll Histopaque (ThermoFisher Scientific, Waltham, MA), according to the manufacturer. CD3+ T-cells were isolated through EasySep magnetic negative isolation (StemCell; Vancouver, BC, Canada) and cultured with 10% AB human serum (Omega Scientific; Tarzana, CA), beta-mercaptoethanol, non-essential amino acids, HEPES, penicillin, streptomycin and gentamicin supplemented X-VIVO media (Corning; Corning, NY).
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