We measured in vitro cell viability and PD-L1 expression using the BD LSR II flow cytometer. Cells were digested into single cells with trypsin EDTA (0.25%), followed by cell viability staining (ThermoFisher Scientific, #L34964) for 20 min at room temperature. After washing with PBS two times, cells were either ready for viability analysis or stained with PD-L1 antibody (ThermoFisher, #12-5982-82) for 20 min under room temperature and washed with PBS three times. In both cases, cells were fixed by 4% polymeric formaldehyde (PFA) after staining and analyzed within 24 h.
Pd l1 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PD-L1 antibody is a laboratory reagent used for detecting the expression of the programmed death-ligand 1 (PD-L1) protein in biological samples. PD-L1 is a key regulator of the immune response and is often expressed by various types of cancer cells. The PD-L1 antibody can be used in research applications to study the role of PD-L1 in immune function and cancer biology.
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Lab products found in correlation
Lsr 2 flow cytometer, by BD (1 mentions)
Transwell support, by Corning (1 mentions)
Cp466722, by Selleck Chemicals (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Anti cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd45 pe cy7, by Thermo Fisher Scientific (1 mentions)
Newborn calf serum nbcs, by Thermo Fisher Scientific (1 mentions)
Bms202, by Selleck Chemicals (1 mentions)
Hematoxylin and eosin h e staining solution, by Merck Group (1 mentions)
Pacific blue anti cd8, by Thermo Fisher Scientific (1 mentions)
Percp cy5.5 anti cd4, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using pd l1 antibody
1
Cell Viability and PD-L1 Evaluation
2
Tumor Cell Invasion Assay with Inhibitors
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Tumor cells (1×104) were seeded into the upper chamber of a Transwell support (Corning Incorporated, Corning, NY, USA) with serum-free media, whereas medium containing 10% FBS was added to the lower chamber. Following incubation for 24 h at 37°C, cells were fixed in methanol and then stained with crystal violet. Positively stained cells in three randomly selected visual fields were counted under a light microscope (Olympus Corporation), magnification, ×20 (inlet, ×100). Each assay was repeated three times. Antibodies used were: JAK inhibitor 1 (420099; EMD Millipore), CP466722 (S2245; Selleck Chemicals), PD-L1 Antibody (62-5982-80, Thermo Fisher Scientific, Inc.).
3
Synthesis of amino-terminated mPEG and reagents
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The amino-terminated poly (ethylene glycol) (mPEG45-NH2) was synthesized through the protocol described in our previous work (Chen et al., 2015 (link)). L-Leucine N-carboxyanhydride (L-Leu NCA) was obtained from Chengdu Enlai Biological Technology Co., Ltd. (Chengdu, China). Penicillin, streptomycin, trypsin-EDTA (0.05% trypsin and 0.02% EDTA) solution, RPMI 1640 medium, and new-born calf serum (NBCS) were bought from Gibco (Grand Island, NY, United States). Toluene, N,N-dimethylformamide (DMF; anhydrous), diethyl ether, elastase, and chymotrypsin were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Hematoxylin and eosin (H&E) staining solution was purchased from Sigma-Aldrich (Shanghai, China). BMS202 was purchased from Selleck Chemicals (United States). PD-L1 antibody and Phospho-VEGF Receptor 2 antibody used for western blot (WB) were purchased from eBioscience (San Diego, CA, United States). PE-cy7-anti-CD45, FITC-anti-CD3, PerCP-cy5.5-anti-CD4, and Pacific Blue-anti-CD8 used for flow cytometry were purchased from eBioscience (San Diego, CA, United States). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai Lengton Bioscience Co., Ltd. (Shanghai, China). All the other chemicals were purchased from Beijing Chemical Industry Group Co., Ltd. (China).
4
PD-L1 Expression Analysis by Flow Cytometry
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Cells were collected and then incubated with PBS (0.5% bovine serum albumin) for 30 mins at room temperature. The cells were probed with PD-L1 antibody (#14-5982-82, eBioscience) at room temperature for 60 mins. Cell suspensions were washed twice in PBS and stained with indicated fluorescent-labeled antibodies for 30 mins. After washing three times with PBS, the cells were analyzed using flow cytometry and data were analyzed using FlowJo X software.
5
Immunohistochemical Analysis of Immune Markers
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The isolated wart lesions and normal skin tissues were fixed with formalin, dehydrated with ethanol and xylene, embedded in paraffin and sliced into 4-μm sections. Then, the tissue sections were deparaffinized in xylene and hydrated through a graded ethanol series. Antigen retrieval was carried out with citrate buffer by heating for 15 min in a microwave. The sections were incubated with H 2 O 2 deionized water and rinsed with PBS. The primary antibodies, including anti-CD86 antibody (1:100, Santa Cruz Biotechnology, CA, USA) and anti-CD163 antibody (1:100, AbD Serotec, Düsseldorf, Germany), PD-1 antibody (1:100, eBioscience, San Diego, CA, USA) and PD-L1 antibody (1:100, eBioscience, San Diego, CA, USA), were added, incubated overnight at 4°C and rinsed with PBS. The secondary antibodies were conjugated with horseradish peroxidase, which was added to the sheep anti-rat/rabbit IgG polymer enzyme marker (MXB, FuJian, China), incubated at 37°C for 2 hours and rinsed with PBS. The sections were dipped into DAB chromogenic agent, washed with water, dehydrated in a graded ethanol series, and then covered with xylene. The immunohistochemistry results were observed.
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