Penicillin

HL-60 and THP-1 cell lines were purchased from Pasteur Institute (Tehran, Iran) and were cultured in Roswell Park Memorial Institute (RPMI) 1640 with 20% heat-inactivated FBS and 1% antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) (Bio idea, Iran). THP-1 is of monocytic origin and representative of AML M-5 (https://en.wikipedia.org/wiki/THP-1_cell_line) while HL-60 is representative of promyelocytic leukemia M-2 and M-3 (https://en.wikipedia.org/wiki/HL60). According to previous studies, THP-1 and HL-60 cell lines express PD-L1 which can be overexpressed by PMA stimulation [9 (link), 10 (link)]. 1 × 10⁶ of each cell line were seeded in each well of the cell culture plate and stimulated with 50 ng/ml phorbol 12-myristate 13 acetate (PMA, Sigma-Aldrich, St Louis, MO, USA) to increase PD-L1 expression. We divided each cell line into two groups including test and control. Both groups were initially stimulated with PMA to express PD-L1. After 24 h of incubation at 37ºC and 5% CO₂, recombinant human PD-1 (CD279)-Fc chimera (carrier-free) (Biolegend, UK) was added to the medium of test group cells in the concentration of 80 ng/ml while the control group cells were not treated. In order to find the effect of PD-L1 stimulation.

Human ESCC YM-1 cell line has already been established in our laboratory at Golestan University of Medical Sciences.^{18 (link)}
Cells were cultured as a monolayer in high glucose DMEM/F12 (BioIdea, CN:1027) supplemented with 10% fetal bovine serum (FBS) (BioIdea, CN:10270-106), 100 IU/mL penicillin and 100 μg/mL streptomycin (BioIdea, CN:1036) at 37°C in a humidified 5% CO2 incubator. The medium was changed every 3 days and cells were passaged to another flask every week.

In this study, Sebacic acid (99%, Merck, Germany), Glycerol (99%, Merck, Germany), Poly Lactic Acid (PLA) (Mw = 200 kDa, Sigma-Aldrich), chloroform (Sigma-Aldrich), Dimethylformamide (DMF) (Merck, Germany), dimethyl thiazole diphenyltetrazolium bromide (MTT, Sigma-Aldrich), sodium citrate (Vacuette, Switzerland), Lipopolysaccharide (LPS, Sigma-Aldrich), dimethyl sulfoxide (DMSO) (Merck, Germany), Dulbecco’s Modified Eagle Medium (DMEM-high and F12-DMEM), fetal bovine serum (FBS), streptomycin, penicillin (Bioidea, Iran), and phosphate buffered saline tablets (PBS, Sigma-Aldrich) were used.

HCT116 and SW48 human colorectal cancer cell lines were purchased from the National Cell Bank, Pasteur Institute of Iran (Tehran, Iran). HCT116 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM F-12 + Glutamax) (Bio-idea, Iran) (Cat No. BI 1027) and SW48 Cell line was cultured in Roswell Park Memorial Institute medium (RPMI 1640+Glutamax) (Bio-idea, Iran) (Cat No. BI 1031), they were supplemented with 10% fetal bovine serum (FBS) (Gibco) (Cat No. 10270-106) and Pen Strep (Penicillin Streptomycin) (100x) (10,000 Units/ml Penicillin, 10,000 μg/ml Streptomycin) (Bio-idea, Iran) (Cat No. BI 1036).