Twelve metastatic melanoma samples were obtained from patients that were not undergoing therapy at the time of sample collection. Patients had undergone a wide range of prior therapies, including surgery, chemotherapy, radiotherapy, immunotherapy, or none of the above. PBLs were obtained by either leukapheresis or venipuncture, prepared over Ficoll-Hypaque gradient (LSM; ICN Biomedicals Inc.), and cryopreserved until analysis. After surgical resection, tumor specimens were processed as previously described (18 (link)). Briefly, tumor specimens were minced, enzymatically digested overnight at room temperature or for several hours at 37°C (RPMI-1640 with l-glutamine [Lonza], 1 mg/ml collagenase IV [Sigma-Aldrich], 30 U/ml DNAse [Genentech], and antibiotics) and the tissue was separated mechanically using gentleMACS (Miltenyi Biotech). Tumor single-cell suspensions were cryopreserved.
Ficoll hypaque gradient lsm
Manufactured by ICN Biomedicals
Ficoll-Hypaque gradient (LSM) is a density gradient medium used for the separation and isolation of mononuclear cells from whole blood, bone marrow, or other tissue samples. It is a sterile, endotoxin-tested solution that allows for the efficient separation of different cell types based on their density differences.
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2 protocols using ficoll hypaque gradient lsm
1
Metastatic Melanoma Sample Preparation
2
Isolation of Tumor-Specific Lymphocytes
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All tumor samples and antigen presenting cells used in this study were obtained from patients enrolled on NCT00068003 and NCT00001823 National Institutes of Health (NIH) clinical protocols, both approved by the Institutional Review Board of the National Cancer Institute. Informed consent was obtained and documented in accordance with the Declaration of Helsinki. Patients were required to be 18 years of age or older, to have measurable metastatic disease, to be of good Eastern Cooperative Oncology Group (ECOG) performance status, and to be free of systemic infections. Metastatic tumor samples were obtained from patients who had been off therapy for at least 1 month at the time of metastasectomy (online supplemental table 1 ). Patients had undergone a wide range of prior therapies relevant to their particular tumor histology including surgery, chemotherapy, radiotherapy, or immunotherapy. Tumor samples were processed as previously described32 (link) using a combination of mechanical separation, enzymatic digestion, and gentleMACS dissociation technology (Miltenyi Biotec); single-cell suspensions were cryopreserved until analysis. Thawed digest samples were rested overnight in media with DNAse (Genentech), without any cytokines, prior to isolation of a lymphocyte-dense fraction over a Ficoll-Hypaque gradient (LSM; ICN Biomedicals).
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