Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: A H&E staining and expression of Ki-67 and N-GSDMD in normal skin tissue samples and skin lesion samples from psoriasis patients were shown. Red arrow indicates N-GSDMD. B Cleaved caspase-1 (green) and PI staining (red) were visualized by immunofluorescence assay. Nucleus was stained by Hoechst ( n = 10). Red arrow indicates PI positive cells. C The cleavage levels of GSDMD and IL-1β were detected in epidermis lysate of normal skin tissue samples and skin lesion samples from psoriasis patients through western blotting assay. D WT mice (C57BL/6NGpt) were stimulated by imiquimod or vaseline, respectively. Skin appearances were presented. Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67. The severity of the lesions was evaluated by PASI scores. The epidermal thickness was measured by Image J software. n = 4. E The co-location of GSDMD and cellular membrane was presented by immunofluorescence assay. Nucleus was stained by Hoechst. Actin filaments was stained by phalloidin ( n = 4). F The cleavage levels of caspase-1, GSDMD and IL-1β were detected in epidermis lysate of mice through western blotting assay. G Flow cytometry was used to determine the level of keratinocyte death. Scale bar represents 100 μm in A . Scale bar represents 500 μm in B . Scale bar represents 50 μm in E . * p < 0.05, ** p < 0.01, *** p < 0.001, WT wild type, GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Expressing, Immunofluorescence, Western Blot, Immunohistochemistry, Software, Membrane, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: A – F WT (C57BL/6NGpt) and Gsdmd −/− mice were stimulated by imiquimod or vaseline, respectively. A Skin appearances were presented. B The severity of the lesions was evaluated by PASI scores (for each group, n = 6). C The epidermal thickness was measured by Image J software (for each group, n = 6). D Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67 ( n = 6). E The levels of loricrin, K5 and MPO were detected by immunohistochemistry study ( n = 6). F Western blotting assay was used to detect the level of cleaved GSDMD and cleaved IL-1β in epidermis lysate of WT or Gsdmd −/− mice ( n = 3). G WT and Gsdmd −/− mice were intradermally injected with recombinant mouse IL-23 (1 μg) in the right ear. The left ear was intradermally injected with vehicle. Skin appearances after injection were presented. H The severity of the lesions was evaluated by PASI scores (for each group, n = 4). I The epidermal thickness was measured by Image J software (for each group, n = 4). J Histological features were analyzed by H&E staining. Scale bar represents 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Software, Staining, Immunohistochemistry, Western Blot, Injection, Recombinant

Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: Control (Krt14 +/+ - Gsdmd flox/flox ) mice and Gsdmd cko (Krt14 Cre/+ - Gsdmd flox/flox ) mice were stimulated by imiquimod or vaseline ( A – H ). A Skin appearances were presented. B The severity of the lesions was evaluated by PASI scores (for each group, n = 5). C The epidermal thickness was measured by ImageJ software (for each group, n = 5). D Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67 ( n = 5). E The efficiency of keratinocyte-specific GSDMD knockout was confirmed by immunofluorescence assay. F Western blotting assay was used to detect the level of cleaved GSDMD and cleaved IL-1β in epidermis lysate of control or Gsdmd cKO mice. G The levels of IL-17 and TNF-α in peripheral blood of imiquimod-stimulated control mice or Gsdmd cKO mice were measured by ELISA ( n = 3). H Ki-67+ and FVS 780+ epidermal cells from imiquimod-stimulated control mice or Gsdmd cKO mice were measured by flowcytometry ( n = 3). Scale bar represents 100 μm in D . Scale bar represents 50 μm in E . * p < 0.05, ** p < 0.01, *** p < 0.001. GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control, Software, Staining, Immunohistochemistry, Knock-Out, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis

doi: 10.1038/s41419-023-06094-3

Figure Lengend Snippet: A GSDMD siRNA or nonsense siRNA was transfected by electroporation into primary human epidermal keratinocytes. The efficiency of knockdown was measured by western blotting assay. siRNA 342 was chosen in the experiments afterwards. B Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay ( n = 3). C PI positive cells (red) and Ki-67 positive cells (green) were determined by immunofluorescence assay ( n = 4). D – F Keratinocytes were treated by M5 for 24 h in presence or absence of DSF (40 μM). D Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay ( n = 3). E PI positive cells (red) and Ki-67 positive cells (green) were determined by immunofluorescence assay ( n = 9). F The secretion of IL-1β, CXCL-2, CCL-20, IL-8 and S100A8/A9 were determined by ELISA ( n = 3). G Keratinocytes were treated by M5 for 24 h in presence or absence of Cu(DTC) 2 . Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay ( n = 3). H , I Imiquimod-induced psoriasis-like dermatitis mice were topically applicated by 1%, 2%, 5% DSF or vehicle. H Skin appearances were presented. Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67. n = 3. I The cleavages of caspase-1, GSDMD and IL-1β in epidermis lysate were detected by western blotting assay ( n = 3). NC: nonsense. Scale bar represents 100 μm. Scale bar represents 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, DSF disulfiram, GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD, siNC nonsense control siRNA, siGSDMD GSDMD siRNA.

Article Snippet: Primary antibodies against cleaved GSDMD (#36425), Ki67 (#12202), caspase-1 (#24232 for mice and #3866 for human), IL-1β (#63124 for mice and #83186 for human) and anti-rabbit IgG HRP-linked secondary antibodies (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Electroporation, Knockdown, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Control

Journal: Clinical and Translational Medicine

Article Title: Neutrophil pyroptosis regulates corneal wound healing and post‐injury neovascularisation

doi: 10.1002/ctm2.1762

Figure Lengend Snippet: GsdmD − / − corneas heal faster and develop less neovascularisation after alkali induced injury. (A) Representative images of fluorescein uptake show that re‐epithelialisation was improved in the corneas of GsdmD − / − mice as compared to WT mice ( n = 5 for GsdmD − / − mice, n = 7 for WT mice). (B) Quantification of fluorescein signal. (C) Representative images of H&E and immunofluorescence staining show that GsdmD − / − corneas have improved re‐epithelialisation with significantly more epithelial cell layers one day after injury. MPO staining indicating that more neutrophils infiltrated the GsdmD −/− mice, as compared to WT corneas. (D) Quantification of corneal epithelial cell layers. (E) At day 10 post alkali injury, GsdmD −/− corneas developed significantly less CNV, as compared to WT corneas. (F) Neovascularisation scores at indicated time points. (G) CD31 staining of flat mount corneas shows that corneas derived from GsdmD − / − mice had less vascularisation than those from WT mice. (H) Quantification of CD31 fluorescent area. Data represent one experiment representative of two independent experiments (A–H). WT, Wild type; MPO, myeloperoxidase; CNV, corneal neovascularisation; H&E, Hematoxylin–Eosin.

Article Snippet: The primary antibodies used for IF staining in this study were anti‐Cleaved GsdmD (N‐terminal) antibody (Cell Signaling Tech, Cat. No. #50928, Discontinued); anti‐IL‐1β antibody (Abcam, Cat. No. ab4722); anti‐MPO antibody (R&D systems, Cat. No. AF3667); FITC anti F4/80 antibody (Biolegned, Cat. No. 123108); anti VEGF Receptor‐1 (Soluble) sflt‐1 antibody (Invitrogen, Cat. No. 36−1100); anti Wnt5A antibody (Thermo Scientific, Cat. No. PA5117496); anti‐CD31 (BD Biosciences, Cat. No. 550274).

Techniques: Immunofluorescence, Staining, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Neutrophil pyroptosis regulates corneal wound healing and post‐injury neovascularisation

doi: 10.1002/ctm2.1762

Figure Lengend Snippet: Neutrophils infiltrate into injured corneas and undergo pyroptosis. (A) Western blotting analysis showed expression of MPO and GsdmD protein in the injured corneas of WT mice at indicated time points after alkali injury. The number of mice used at each time point ranges from 5 to 7. (B) Representative immunofluorescent images of flat mount corneas were stained with cleaved GsdmD (GsdmD‐N), F4/80, and MPO. Co‐localisation of MPO and GsdmD‐N was detected. The corneas were stained 24 h after injury. (C) WT mice receiving Gr‐1 antibody treatments had a dramatic reduction in MPO and GsdmD‐N expression at 24 h after alkali induced injury ( n = 4 for each group). Data represent one experiment representative of two independent experiments (A–C). WT, Wild type; MPO, myeloperoxidase.

Article Snippet: The primary antibodies used for IF staining in this study were anti‐Cleaved GsdmD (N‐terminal) antibody (Cell Signaling Tech, Cat. No. #50928, Discontinued); anti‐IL‐1β antibody (Abcam, Cat. No. ab4722); anti‐MPO antibody (R&D systems, Cat. No. AF3667); FITC anti F4/80 antibody (Biolegned, Cat. No. 123108); anti VEGF Receptor‐1 (Soluble) sflt‐1 antibody (Invitrogen, Cat. No. 36−1100); anti Wnt5A antibody (Thermo Scientific, Cat. No. PA5117496); anti‐CD31 (BD Biosciences, Cat. No. 550274).

Techniques: Western Blot, Expressing, Staining

Journal: Clinical and Translational Medicine

Article Title: Neutrophil pyroptosis regulates corneal wound healing and post‐injury neovascularisation

doi: 10.1002/ctm2.1762

Figure Lengend Snippet: Pyroptotic neutrophils inhibit corneal epithelial cell migration and reduce monolayer integrity. (A) Corneas from indicated treatments were incubated with corneal epithelial cells. Scratch wounds were induced and representative images were taken at 0 and 6 h after scratch wound. (B) Quantification of scratch wounds. (C) Co‐culture of isolated neutrophils derived from WT mice, but not Gsdmd−/− mice, reduced migration of corneal epithelial cells; (D) quantification of migration rates. (E) Co‐culture of WT neutrophils with corneal epithelial cells disrupted epithelial integrity, as measured by TEER. Data represent one experiment representative of three independent experiments (A–E). WT, wild type; TEER, transepithelial electrical resistance.

Article Snippet: The primary antibodies used for IF staining in this study were anti‐Cleaved GsdmD (N‐terminal) antibody (Cell Signaling Tech, Cat. No. #50928, Discontinued); anti‐IL‐1β antibody (Abcam, Cat. No. ab4722); anti‐MPO antibody (R&D systems, Cat. No. AF3667); FITC anti F4/80 antibody (Biolegned, Cat. No. 123108); anti VEGF Receptor‐1 (Soluble) sflt‐1 antibody (Invitrogen, Cat. No. 36−1100); anti Wnt5A antibody (Thermo Scientific, Cat. No. PA5117496); anti‐CD31 (BD Biosciences, Cat. No. 550274).

Techniques: Migration, Incubation, Co-Culture Assay, Isolation, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Neutrophil pyroptosis regulates corneal wound healing and post‐injury neovascularisation

doi: 10.1002/ctm2.1762

Figure Lengend Snippet: Wnt5A expression is localised to the limbus of uninjured corneas and in newly regenerated corneal epithelium following injury. (A) mRNA expression of several known wound healing markers in corneas derived from WT and GsdmD −/− mice at 24 h after injury ( n = 5 for each group). (B) Immunofluorescent staining of mouse eyes shows Wnt5A is highly expressed in the newly differentiated corneal epithelial cells after injury. (C) Flat mount staining of mouse corneas demonstrates that Wnt5A is mainly expressed in limbus before injury. After alkali injury, Wnt5A is expressed in newly generated epithelial cells. Macrophages (F4/80) and neutrophils (MPO) were also observed after injury. Data represent one experiment representative of three independent experiments (A–C).

Article Snippet: The primary antibodies used for IF staining in this study were anti‐Cleaved GsdmD (N‐terminal) antibody (Cell Signaling Tech, Cat. No. #50928, Discontinued); anti‐IL‐1β antibody (Abcam, Cat. No. ab4722); anti‐MPO antibody (R&D systems, Cat. No. AF3667); FITC anti F4/80 antibody (Biolegned, Cat. No. 123108); anti VEGF Receptor‐1 (Soluble) sflt‐1 antibody (Invitrogen, Cat. No. 36−1100); anti Wnt5A antibody (Thermo Scientific, Cat. No. PA5117496); anti‐CD31 (BD Biosciences, Cat. No. 550274).

Techniques: Expressing, Derivative Assay, Staining, Generated

Journal: Clinical and Translational Medicine

Article Title: Neutrophil pyroptosis regulates corneal wound healing and post‐injury neovascularisation

doi: 10.1002/ctm2.1762

Figure Lengend Snippet: sflt‐1 is highly expressed in newly generated corneal epithelium following injury. (A) mRNA expression of VEGFa, VEGFR1, VEGFR2 and sflt‐1 was quantified using real‐time RT‐PCR from WT and GsdmD −/− corneas 24 h after injury ( n = 5 for each group). (B) Expression of sllt‐1, as observed using immunofluorescence, was observed in corneal epithelial and stromal cells before injury and expression of sflt‐1 in epithelial cells was enhanced after corneal injury. Data represent one experiment representative of three independent experiments (A,B).

Article Snippet: The primary antibodies used for IF staining in this study were anti‐Cleaved GsdmD (N‐terminal) antibody (Cell Signaling Tech, Cat. No. #50928, Discontinued); anti‐IL‐1β antibody (Abcam, Cat. No. ab4722); anti‐MPO antibody (R&D systems, Cat. No. AF3667); FITC anti F4/80 antibody (Biolegned, Cat. No. 123108); anti VEGF Receptor‐1 (Soluble) sflt‐1 antibody (Invitrogen, Cat. No. 36−1100); anti Wnt5A antibody (Thermo Scientific, Cat. No. PA5117496); anti‐CD31 (BD Biosciences, Cat. No. 550274).

Techniques: Generated, Expressing, Quantitative RT-PCR, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Neutrophil pyroptosis regulates corneal wound healing and post‐injury neovascularisation

doi: 10.1002/ctm2.1762

Figure Lengend Snippet: Inhibition of pyroptosis enhances corneal wound healing and inhibits post‐injury corneal neovascularisation. (A) Bone marrow cells from WT and GsdmD −/− mice were transplanted to irradiated WT mice. Mice that received GsdmD −/− bone marrow cells had improved re‐epithelialisation ( n = 5 for WT‐to‐WT mice, n = 4 for GsdmD −/− ‐to‐WT mice) (quantified in B). (C) Dual antibodies treatment to deplete neutrophils ( n = 5 for each group) or (E) disulfiram treatment to inhibit pyroptosis also promoted corneal re‐epithelialisation ( n = 6 for disulfiram treated mice and n = 4 for sesame oil treated mice) (quantified in D and F). (G) CD31 staining of corneal flat mounts demonstrates that disulfiram treatment could significantly reduce neovascularisation after corneal injury (quantified in H). Data represent one experiment representative of two independent experiments (A–H). WT, Wild type.

Article Snippet: The primary antibodies used for IF staining in this study were anti‐Cleaved GsdmD (N‐terminal) antibody (Cell Signaling Tech, Cat. No. #50928, Discontinued); anti‐IL‐1β antibody (Abcam, Cat. No. ab4722); anti‐MPO antibody (R&D systems, Cat. No. AF3667); FITC anti F4/80 antibody (Biolegned, Cat. No. 123108); anti VEGF Receptor‐1 (Soluble) sflt‐1 antibody (Invitrogen, Cat. No. 36−1100); anti Wnt5A antibody (Thermo Scientific, Cat. No. PA5117496); anti‐CD31 (BD Biosciences, Cat. No. 550274).

Techniques: Inhibition, Irradiation, Staining

Journal: Clinical and Translational Medicine

Article Title: Neutrophil pyroptosis regulates corneal wound healing and post‐injury neovascularisation

doi: 10.1002/ctm2.1762

Figure Lengend Snippet: Enhanced corneal wound healing and reduced CNV in GsdmD myeloid conditional knockout mice. Representative images demonstrating fluorescein uptake illustrate enhanced re‐epithelialisation in the corneas of Gsdmd fl/fl : Lysm Cre +/− mice compared to those of Gsdmd fl/fl : Lysm Cre −/− mice ( n = 5 for each group). (B) Quantification of fluorescein assay. (C) Representative images of ocular photographs depict corneal fibrosis and neovascularisation at both pre‐injury and 14 days post‐injury. (D) Opacity scores at indicated time points. (E) Neovascularisation scores at indicated time points. (F) Representative images of CD31 staining of flat mount corneas. (G) Quantification of CD31 signal area. Data represent one experiment representative of two independent experiments (A–E). CNV, Corneal neovascularisation.

Article Snippet: The primary antibodies used for IF staining in this study were anti‐Cleaved GsdmD (N‐terminal) antibody (Cell Signaling Tech, Cat. No. #50928, Discontinued); anti‐IL‐1β antibody (Abcam, Cat. No. ab4722); anti‐MPO antibody (R&D systems, Cat. No. AF3667); FITC anti F4/80 antibody (Biolegned, Cat. No. 123108); anti VEGF Receptor‐1 (Soluble) sflt‐1 antibody (Invitrogen, Cat. No. 36−1100); anti Wnt5A antibody (Thermo Scientific, Cat. No. PA5117496); anti‐CD31 (BD Biosciences, Cat. No. 550274).

Techniques: Knock-Out, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Caspase-8-and Gasdermin D (GSDMD)-Dependent PANoptosis Participate in the Seasonal Atrophy of Scented Glands in Male Muskrats

doi: 10.3390/ani14223194

Figure Lengend Snippet: Real-time quantitative PCR was used to detect the mRNA expression levels of the following genes in muskrats’ scented glands during the breeding and non-breeding seasons. ( a ) TNFR1, TRADD, FADD, Caspase-8, BAX, and BCL2. ( b ) NLRP3, ASC, Caspase-1, GSDMD, and IL-1β. ( c ) TAK1, RIPK1, Caspase-8, GSDMD, and FADD. ( d , e ) Also shown are the protein expression results of GSDMD and Caspase-8. ( f – i ) The grayscale analysis of GSDMD, GSDMD-N, Caspase-8 p18, and Caspase-8 p43. B—breeding season; NB—non-breeding season. The error bars represent the means ± SEM ( n = 3, each stage). * Statistical significance (* p < 0.05; ** p < 0.01).

Article Snippet: The sections were blocked at room temperature with BSA for 1 h and then incubated overnight at 4 °C with the following primary antibodies: Cleaved GSDMD (N Terminal) Rabbit mAb (Abclonal, A22523, Boston, MA, USA) and Caspase 8/p43/p18 Monoclonal antibody (Proteintech, 66093-1-Ig, Wuhan, China).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Caspase-8-and Gasdermin D (GSDMD)-Dependent PANoptosis Participate in the Seasonal Atrophy of Scented Glands in Male Muskrats

doi: 10.3390/ani14223194

Figure Lengend Snippet: Immunofluorescence results in muskrats’ scented glands during the breeding ( a – d ) and non-breeding seasons ( e – h ). The green ( a , e ) and red ( b , f ) fluorescence signals represent GSDMD and Caspase-8, respectively. Scale bar = 100 μm.

Article Snippet: The sections were blocked at room temperature with BSA for 1 h and then incubated overnight at 4 °C with the following primary antibodies: Cleaved GSDMD (N Terminal) Rabbit mAb (Abclonal, A22523, Boston, MA, USA) and Caspase 8/p43/p18 Monoclonal antibody (Proteintech, 66093-1-Ig, Wuhan, China).

Techniques: Immunofluorescence, Fluorescence

Journal: Mucosal Immunology

Article Title: Epithelial GPR35 protects from Citrobacter rodentium infection by preserving goblet cells and mucosal barrier integrity

doi: 10.1038/s41385-022-00494-y

Figure Lengend Snippet: a Protein expression of cleaved GSDMD, cleaved caspase-11 and cleaved caspase-1 in crypt samples isolated from the proximal colon obtained from Gpr35 f/f Vil + ( n = 3) and Gpr35 wt co-housed littermates ( n = 3). b Densitometry analysis of ( a ). c Protein expression of cleaved GSDMD and cleaved caspase-11 in sorted goblet cells and colonocytes obtained from Gpr35 f/f Vil + ( n = 3) and Gpr35 wt co-housed littermates ( n = 3). d Densitometry analysis of ( c ). e Flow cytometry assessment of cell apoptosis (TUNEL) percentage in goblet cells obtained from the proximal colon of Gpr35 f/f Vil + (red histogram) and Gpr35 wt (gray histogram) co-housed littermates. Numbers in histograms indicate the percentage of apoptotic goblet cells. The blue histogram represents the negative control of the assay. f Protein expression of cleaved GSDMD and cleaved caspase-11 in proximal colon explant obtained from Gpr35 wt mice treated with ML194 at 10 μM for 3 h. g Densitometry analysis of ( f ). h Protein expression of cleaved GSDMD and cleaved caspase-11 in proximal colon explants obtained from Gpr35 wt mice treated with either Zaprinast or LPA at 10 μM for 1.5 h followed by OMVs treatment at 10 μg for 1.5 h. i Densitometry analysis of ( h ). Each dot represents one animal with medians. Data are represented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 by Mann–Whitney test.

Article Snippet: The nitrocellulose membrane was then incubated overnight with the following primary antibodies: cleaved GSDMD (Cell Signaling Technology, #50928), cleaved caspase-11 (Abcam, #ab180673), cleaved caspase-1 (Invitrogen #AB 5B10), and β-actin (BD Biosciences #612656) at 1:1000 dilution.

Techniques: Expressing, Isolation, Flow Cytometry, TUNEL Assay, Negative Control, MANN-WHITNEY