1x flow cytometry staining buffer  (Thermo Fisher)

Journal: Cancer Immunology Research

Article Title: Cannabidiol Enhances Atezolizumab Efficacy by Upregulating PD-L1 Expression via the cGAS–STING Pathway in Triple-Negative Breast Cancer Cells

doi: 10.1158/2326-6066.CIR-23-0902

Figure Lengend Snippet: CBD upregulates PD-L1 expression in TNBC cells. A and B, RNA-seq analysis of PD-L1 expression in different breast cancer subtypes. A, TNBC ( n = 584), HER2 ( n = 343), luminal A ( n = 1877), and luminal B ( n = 1,617), as well as TNBC and non-TNBC subtypes. B, TNBC ( n = 293) and non-TNBC subtypes ( n = 3,887). C, Endogenous protein levels of PD-L1 in various breast cancer cell lines and normal breast cell lines were determined by Western blot analysis. β-actin was used as a loading control. D, Human TNBC cell lines (MDA-MB-231 and BT20) were treated with increasing concentrations of CBD (0, 1, 2.5, and 5 μmol/L) for 24 hours. Protein levels of PD-L1 were determined by Western blot analysis. β-actin was used as a loading control. E, MDA-MB-231 and BT20 cells were treated with increasing concentrations of CBD (0, 1, 2.5, and 5 μmol/L) for 24 hours. Relative PD-L1 mRNA expression levels were measured by qRT-PCR. F, Flow cytometry analysis showing the expression of cell surface PD-L1 in TNBC cells. The relative MFI of PD-L1 expression is shown. Data are shown as the mean ± SD and analyzed by unpaired Student t test. **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were resuspended in 200 μL 1× flow cytometry staining buffer (FC001, R&D Systems) and incubated with PE-conjugated antibodies against PD-L1 or isotype control for 30 minutes at room temperature in the dark.

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Control, Quantitative RT-PCR, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: Cannabidiol Enhances Atezolizumab Efficacy by Upregulating PD-L1 Expression via the cGAS–STING Pathway in Triple-Negative Breast Cancer Cells

doi: 10.1158/2326-6066.CIR-23-0902

Figure Lengend Snippet: Combination treatment of CBD and atezolizumab triggers anticancer immune response. A, MDA-MB-231 and BT20 cells were preincubated with 2.5 μmol/L CBD and 50 μg/mL atezolizumab for 1 hour and subsequently cocultured with activated Jurkat T cells at a cancer to T-cell ratio of 1:10 in the presence of a stimulator (anti-CD3/CD28) for additional 24 hours. Luciferase activity was measured using a luciferase system and microplate reader. B and C, MDA-MB-231 and BT20 cells were preincubated with 2.5 μmol/L CBD and 50 μg/mL atezolizumab for 1 hour and subsequently cocultured with activated Jurkat T cells or human PBMCs in the presence of a stimulator (anti-CD3/CD28) for additional 24 hours. Levels of IL2 ( B ) and IFNγ ( C ) were measured by ELISA assay. D–F, Human PBMC-mediated tumor-killing assay. MDA-MB-231 cells were pretreated with or without 2.5 μmol/L CBD and 50 μg/mL atezolizumab for 1 hour and cocultured with human PBMCs at the indicated effector and target cell ratio of 10:1 (effector: PBMCs; target: TNBC cells) for additional 24 hours. Human PBMCs and cell debris were washed and removed with PBS. Living tumor cells were visualized by crystal violet staining and washed with acetic acid solution. Bar graph representing the proportion of death cells is shown ( D ). The expression of cleaved (c)-PARP was determined by Western blot analysis ( E ). β-actin was used as a loading control ( F ). The proportion of apoptotic cell death in tumor cells was estimated by annexin V/PI staining by flow cytometry analysis. Data are shown as the mean ± SD and analyzed by unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were resuspended in 200 μL 1× flow cytometry staining buffer (FC001, R&D Systems) and incubated with PE-conjugated antibodies against PD-L1 or isotype control for 30 minutes at room temperature in the dark.

Techniques: Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Western Blot, Control, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: Cannabidiol Enhances Atezolizumab Efficacy by Upregulating PD-L1 Expression via the cGAS–STING Pathway in Triple-Negative Breast Cancer Cells

doi: 10.1158/2326-6066.CIR-23-0902

Figure Lengend Snippet: CBD promotes PD-L1 upregulation via activation of the cGAS–STING pathway in TNBC cells. A, Correlation between IRF3 and PD-L1 expression was evaluated by RNA-sequence analysis by BC-GenExMiner v4.9 web online tool. B, Western blot analysis of the cGAS–STING pathway in MDA-MB-231 cells after treatment with CBD. C, MDA-MB-231 cells were transfected with cGAS siRNA. Transfected cells were treated with 2.5 μmol/L CBD for 24 hours. Protein expression was detected by Western blot analysis. β-actin was used as a loading control. D and E, MDA-MB-231 cells were treated with 10 nmol/L dimeric amidobenzimidazole (diABZI) STING agonist for 24 hours. F and G, MDA-MB-231 cells were pretreated with 1 μg/mL H151 STING antagonist for 1 hour. After pretreatment, the cells were treated with 2.5 μmol/L CBD for 24 hours. Protein expression was detected by Western blot analysis ( D and F ). β-actin was used as a loading control. Expression of cell surface PD-L1 was determined by flow cytometry analysis ( E and G ). The relative MFI for PD-L1 expression levels is shown. Data are shown as the mean ± SD and analyzed by unpaired Student t test. ***, P < 0.001.

Article Snippet: Cells were resuspended in 200 μL 1× flow cytometry staining buffer (FC001, R&D Systems) and incubated with PE-conjugated antibodies against PD-L1 or isotype control for 30 minutes at room temperature in the dark.

Techniques: Activation Assay, Expressing, Sequencing, Western Blot, Transfection, Control, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: Cannabidiol Enhances Atezolizumab Efficacy by Upregulating PD-L1 Expression via the cGAS–STING Pathway in Triple-Negative Breast Cancer Cells

doi: 10.1158/2326-6066.CIR-23-0902

Figure Lengend Snippet: IRF3 binds directly to the PD-L1 gene promoter region in TNBC cells. A, A schematic representation of the PD-L1 gene promoter region. Three possible IRF3-binding site sequences, named BS1, BS2, and BS3, were indicated in red upstream of transcription start site in the PD- L1 gene. B, Sequence of the PD-L1 promoter region. Three possible IRF3-binding site sequences on the PD-L1 promoter region are shown in red. C, The sequence of the IRF3-binding motif. D, Products of ChIP-PCR in the input, IgG, IRF3, and water (DW) groups were analyzed by agarose gel electrophoresis. PCR was performed with specific primers of IRF3-binding sites. E–G, MDA-MB-231 cells were transfected with control and IRF3 siRNA. Transfected cells were treated with 2.5 μmol/L CBD for 24 hours. The mRNA ( E ), protein ( F ), and expression of cell surface PD-L1 ( G ) were subjected by qRT-PCR, Western blot analysis, and flow cytometry analysis. The relative MFI for PD-L1 expression level is shown. Data are shown as the mean ± SD and analyzed by unpaired Student t test. ***, P < 0.001.

Article Snippet: Cells were resuspended in 200 μL 1× flow cytometry staining buffer (FC001, R&D Systems) and incubated with PE-conjugated antibodies against PD-L1 or isotype control for 30 minutes at room temperature in the dark.

Techniques: Binding Assay, Sequencing, Agarose Gel Electrophoresis, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: Cannabidiol Enhances Atezolizumab Efficacy by Upregulating PD-L1 Expression via the cGAS–STING Pathway in Triple-Negative Breast Cancer Cells

doi: 10.1158/2326-6066.CIR-23-0902

Figure Lengend Snippet: Synergistic effect of CBD and anti–PD-L1 in in vivo experiments. A, The tumor volume of each group was measured every 2 days ( n = 6). B, Tumor images were determined after the mice were sacrificed. C, Flow cytometry analysis was performed to estimate PD-L1 expression in tumor cells from each group ( n = 6). The relative MFI of PD-L1 expression is shown. D–F, IHC staining of PD-L1 ( D ), CD8 + T cells ( E ), and granzyme B ( F ) in tumor sections from each group. Quantifications of each group ( n = 6). Data are shown as the mean ± SD and analyzed by unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were resuspended in 200 μL 1× flow cytometry staining buffer (FC001, R&D Systems) and incubated with PE-conjugated antibodies against PD-L1 or isotype control for 30 minutes at room temperature in the dark.

Techniques: In Vivo, Flow Cytometry, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Erythropoietin decreases apoptosis and promotes Schwann cell repair and phagocytosis following nerve crush injury in mice

doi: 10.1101/2025.01.22.634402

Figure Lengend Snippet: A, B Representative IHC images and quantitative results of repair SCs (anti-p75-NTR staining) phagocytosis of myelin debris (anti-MPZ staining) in saline and EPO treated nerve tissues on post-SNCI days 3, 5, and 7. n = 5/ group. C, D Representative IF images and quantitative results of repair SCs (phalloidin staining) phagocytosis of myelin debris (PKH26 staining) following 24h EPO (10IU/ mL) treatment under LPS (500ng/ mL) stress conditions. n = 3/ group. E, F Flow cytometry images and quantitative results of repair SCs (p75-NTR positive cells) phagocytosis of myelin debris (PKH26 staining) following 24h EPO (10IU/ mL) treatment under LPS (500ng/ mL) stress conditions. n = 3/ group. Data are represented as mean□±□SEM. The statistical significance is indicated by asterisks (**P < 0.0021, ***P < 0.0002, and ****P□<□0.0001 vs. saline group) and compared using two-tailed, unpaired t-tests or ordinary one-way ANOVA.

Article Snippet: Next, cells were resuspended in 1X flow cytometry staining buffer (# FC001, R&D) and were stained with p75-NTR (1:100, # BS-0161R, Bios) conjugated antibody for 30 min. After staining, the cells were resuspended in a staining buffer.

Techniques: Staining, Saline, Flow Cytometry, Two Tailed Test