Ficoll isopaque

The melanoma cell line, Mel526, and breast carcinoma cell line, MDA-MB-231, were kindly provided by Xiangxue Life Sciences Research Center (Guangzhou, China). NY-ESO-1^−/− Mel526 was constructed by CRISPR/Cas9 system, and NY-ESO-1 CRISPR/Cas9 KO plasmids were purchased from Santa Cruz Biotechnology (SC-418340, Dallas, USA). The lentivirus packaging cell line HEK-293T (RRID: CVCL_0063) was purchased from the ATCC (Rockville, USA) and cultured in DMEM containing 10% FBS.
Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood samples of healthy volunteers by density gradient centrifugation using Ficoll-Isopaque (Axis shield) which reported before [21 (link)]. An informed consent was obtained from the volunteers who donated blood samples. PBMCs were used to make peripheral blood lymphocytes (PBLs), which were then stimulated with Human T-Activator CD3/CD28 Dynabeads (Life Technologies, Carlsba, USA) in RPMI 1640 medium with 10% fetal bovine serum and 100 IU of recombinant human IL-2 [22 (link)]. STING inhibitor and agonist, H151 and diABZI, were purchased from InvivoGen (Hong Kong, China). T cells and tumor cells were treated for 3 h separately, completely washed off with PBS twice, and then co-cultured.

PBMCs were freshly isolated from buffy coats using density gradient centrifugation on Ficoll-Isopaque (Lymphoprep® Axis-Shield, Norway), according to the manufacturer's protocol. Human CD3⁺ T cells were isolated by negative selection (Miltenyi Biotec; Human Pan T Cell Isolation Kit) according to the manufacturer's instructions. CD3⁺ T cells (purity >95%) were then stained with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) and activated using anti-CD3/CD28 microbeads (Miltenyi Biotec) for 5 days. Using flow cytometry (BD Fortessa LSR-II), proliferation of T cells was assessed in the presence or absence of MSC. All antibodies used for T cell staining are presented in Table 1. Depending on conditions, MSC were treated for 1 h with plasma or heat-inactivated plasma prior to co-culture with T cells. Data were analyzed using FlowJo software (Ashland, OH).