cellrox® deep red flow cytometry assay kit  (Thermo Fisher)

Journal: Advanced Science

Article Title: 3‐D Sustained‐Release Culture Carrier Alleviates Rat Intervertebral Disc Degeneration by Targeting STING in Transplanted Skeletal Stem Cells

doi: 10.1002/advs.202410151

Figure Lengend Snippet: SSCs pretreated with PH effectively delay IVDD. A) Flow chart of SSC extraction from the costal cartilage of rats. Costal cartilage was obtained from 1–2‐week‐old rats and cut into 1–2 mm 3 samples. A mixture of 2 mg mL −1 collagenase 2 and collagenase 4 was added for digestion for 1–2 h and then filtered through a 40 µm filter. Finally, the SSCs from the filtered cell suspension were sorted by flow cytometry and cultured in the incubator. B) Sorting and identification of the costal cartilage‐derived stem cells by flow cytometry, labeled CD90 − CD45 − CD105 − ENPEP − Tie2 − OX‐83 − CD51 + . C) Identification of chondrogenic and osteogenic potential and lack of adipogenic potential of SSCs using Alcian blue (left), alizarin red (middle), and Oil Red O (right) staining. D) Sectioned grafts stained with Movat's pentachrome. E) Treatment of IVDD after SSCs were cultured under normoxia or PH preconditioning. F, G) X‐ray and statistical results of the height of IVDs in the NC group ( n = 3), puncture group ( n = 3), puncture + SSCs group ( n = 3), or puncture + PH‐preconditioned SSCs group ( n = 3). H, I) MRI and statistical results of Pfirrmann grades. J) Safranin O‐fast green staining in NC, puncture, puncture + SSCs, or puncture + PH‐preconditioned SSCs IVDs. PH: progressive hypoxia. NP: nucleus pulposus; AF: annulus fibrosus; SSCs: skeletal stem cells; NPLC: nuclear pulposus‐like cell. Data in G, I) are presented as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, no significant difference by one‐way analysis of variance (ANOVA) test.

Article Snippet: C11‐BODIPY 581/591 (cat. no. D3861; Thermo Fisher Scientific) or a CellROX Deep Red Flow Cytometry Assay Kit (cat. no. C10491, Thermo Fisher Scientific) was used to analyze ROS and lipid peroxidation levels.

Techniques: Extraction, Suspension, Flow Cytometry, Cell Culture, Derivative Assay, Labeling, Staining

Journal: Advanced Science

Article Title: 3‐D Sustained‐Release Culture Carrier Alleviates Rat Intervertebral Disc Degeneration by Targeting STING in Transplanted Skeletal Stem Cells

doi: 10.1002/advs.202410151

Figure Lengend Snippet: PH preconditioning improves the viability of SSCs after injection into IVDs. A) After culturing under normoxia or PH preconditioning, DIR‐labeled SSCs were injected into the IVDs of rats and imaged in vivo at different time points ( n = 6). The six rats were divided into two groups of three. B) Flowchart of SSC counting and ferroptosis detection in the IVDs. Ten rats were divided into two groups of five. SSCs were cultured under normoxia or PH preconditioning and then stained with Calcein‐AM and injected into the IVDs. Five or six IVDs were selected from the tail of each rat for SSC injection. After 2–3 day, the rats were euthanized, the IVDs were opened, and the tissues and cells in the nucleus pulposus region were gently dissociated with PBS and collected in PBS solution. After gentle shaking and filtration through a 40 µm filter, a single SSC suspension was obtained. Cell precipitate containing a large number of SSCs was obtained by centrifugation, and then flow cytometry, western blotting, and transmission electron microscopy (TEM) were performed. C) Flow cytometry results for SSC survival after entering the IVD. ≈15 000 Calcein‐AM positive SSCs were injected into the IVDs. After 2–3 day, all cells in the IVDs were obtained and filtered through a 40 µm filter. Cell pellets was obtained by centrifugation and resuspended in 500 µL PBS. Flow cytometry was used to collect 50000 cells and calculate the volume V of the remaining cell suspension. The number N of Calcein‐AM‐positive SSCs was calculated based on the positive ratio R . Thus, the number of remained Calcein‐AM‐positive SSCs in the IVD after 2–3 days was calculated as N = 50 000 × R ×500/(500– V ). D, E) Statistical analysis of the number and proportion of remaining Calcein‐AM‐positive SSCs in the IVDs. F) Western blotting and quantitative protein levels of ACSL4 in SSCs. The “Normoxic SSCs in vitro” group was derived from the SSCs cultured under normoxic conditions in vitro. The “Normoxic SSCs in IVD” group was derived from SSCs injected into the intervertebral disc after normoxic culture in vitro. The “PH preconditioned SSCs in IVD” group was derived from SSCs injected into the intervertebral disc after PH preconditioning in vitro. G) TEM detection of mitochondrial damage in different SSC treatment groups. H) Diagram showing the preparation of tissue slices via IVD transection. I) Detection of ROS in transected IVD tissue slices after injection with phosphate‐buffered saline (NC), normoxic SSCs, or PH‐conditioned SSCs. NC: Normal Control. Data in D‐E) are presented as the mean ± SD, *** p < 0.001 by two‐tailed Student’s t‐test. Data in F) are presented as the mean ± SD, * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one‐way ANOVA.

Article Snippet: C11‐BODIPY 581/591 (cat. no. D3861; Thermo Fisher Scientific) or a CellROX Deep Red Flow Cytometry Assay Kit (cat. no. C10491, Thermo Fisher Scientific) was used to analyze ROS and lipid peroxidation levels.

Techniques: Injection, Labeling, In Vivo, Cell Culture, Staining, Gentle, Filtration, Suspension, Centrifugation, Flow Cytometry, Western Blot, Transmission Assay, Electron Microscopy, In Vitro, Derivative Assay, Saline, Control, Two Tailed Test

Journal: Advanced Science

Article Title: 3‐D Sustained‐Release Culture Carrier Alleviates Rat Intervertebral Disc Degeneration by Targeting STING in Transplanted Skeletal Stem Cells

doi: 10.1002/advs.202410151

Figure Lengend Snippet: cGAS/STING aggravates ferroptosis in SSCs via the HIF‐1α/mitophagy axis. A–C) GO, KEGG, and GSEA analyses of genes in SSCs under hypoxia and normoxia. D–F) Western blotting and quantified protein levels of ACSL4, GPX4, HIF‐1α, STING, and cGAS in SSCs treated with hypoxia or normoxia. G, H) Representative Western blotting and mRNA levels of ACSL4, STING, cGAS, and HIF‐1α in SSCs in the NC, hypoxia, and hypoxia + C‐176 (5 µ m ) groups. I) Representative Western blotting of ACSL4 and HIF‐1α in SSCs in the NC, hypoxia, erastin, and hypoxia + erastin (10 µ m ) groups. J) GSEA analysis of SSCs cultured under normoxia and hypoxia. K) Venn diagram of ferroptosis driver genes, mitophagy genes, and HlF‐1α pathway genes. L, M) Western blotting and quantitative protein levels of FOXO3 and HIF‐1α in SSCs in the NC, Lenti‐HIF‐1α, and Lenti‐HIF‐1α + KC7F2 groups. N) Venn diagram of mitophagy genes and FOXO3 signaling pathway genes. O, P) Western blotting and quantitative protein levels of LC3II/I, PARKIN, PINK1, and FOXO3 in SSCs treated with hypoxia, hypoxia + Lenti‐FOXO3, or hypoxia + si‐FOXO3. Q, R) Western blotting and quantitative protein levels of ACSL4, PARKIN, and P62 in SSCs in the NC, hypoxia, and hypoxia + Olaparib groups. S, T) Western blotting and quantitative protein levels of HIF‐1α, ACSL4, Pink1, and STING in SSCs in the NC, hypoxia, hypoxia + DMXAA, and hypoxia + DMXAA + NAC groups. U) Detection of mitochondrial damage in different SSC treatment groups using TEM. V) CellROX Deep Red staining for lipid peroxidation in SSCs in the NC, hypoxia, hypoxia + DMXAA, and hypoxia + DMXAA + NAC groups. W) C11‐BODIPY staining for ROS levels in different SSC treatment groups. Data in F) are presented as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001 by two‐tailed Student's t ‐test. Data in H, M, P, R, T, V, W) are presented as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significant difference by one‐way ANOVA.

Article Snippet: C11‐BODIPY 581/591 (cat. no. D3861; Thermo Fisher Scientific) or a CellROX Deep Red Flow Cytometry Assay Kit (cat. no. C10491, Thermo Fisher Scientific) was used to analyze ROS and lipid peroxidation levels.

Techniques: Western Blot, Cell Culture, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: 3‐D Sustained‐Release Culture Carrier Alleviates Rat Intervertebral Disc Degeneration by Targeting STING in Transplanted Skeletal Stem Cells

doi: 10.1002/advs.202410151

Figure Lengend Snippet: PH reduces ROS levels and promotes the differentiation of SSCs into NPLCs by downregulating STING expression. A) Heatmap analysis of SSCs cultured under normoxia, hypoxia, or PH preconditioning. B) KEGG analysis of SSCs cultured under hypoxia or PH preconditioning. C) GSEA analysis of SSCs cultured under NC, hypoxia, or PH preconditioning. D, E) Western blotting and quantitative protein levels of ACSL4, STING, ACAN, and COL2 in SSCs treated with NC, hypoxia, or PH preconditioning. F) Cell viability and levels of GSH, MDA, and intracellular iron ions detected in SSCs treated with NC, hypoxia, or PH preconditioning. G) CellROX Deep Red staining in SSCs treated with TBHP and different concentrations of NAC (0, 40, 80, 120, or 200 µ m ). H) Mean fluorescence intensity (MFI) of ROS and mRNA levels and statistical analysis in SSCs treated with TBHP and different concentrations of NAC (0, 40, 80, 120, or 200 µ m ) compared with NC. I) Immunofluorescence staining of COL2/GPX4 in SSCs treated with TBHP and different concentrations of NAC (0, 40, 80, 120, or 200 µ m ). J, K) Western blotting and quantitative protein levels of TCF‐4, β‐Catenin, and COL2 in SSCs treated with TBHP (60 µ m ) and NAC (0, 40, 80, 120, or 200 µ m ). L, M) Western blotting and quantitative protein levels of ACSL4, TCF‐4, COL2, and ACAN in SSCs in the NC, TBHP, TBHP + C‐176, and TBHP + DMXAA groups. N) Schematic of the cGAS/STING regulation of ferroptosis and differentiation under hypoxia or PH. Data in E, F, H, K, M) are presented as the mean ± SD, *p and # p < 0.05, ** p and ## p < 0.01, *** p and ### p < 0.001, **** p and #### p < 0.0001, ns, no significant difference by one‐way ANOVA.

Article Snippet: C11‐BODIPY 581/591 (cat. no. D3861; Thermo Fisher Scientific) or a CellROX Deep Red Flow Cytometry Assay Kit (cat. no. C10491, Thermo Fisher Scientific) was used to analyze ROS and lipid peroxidation levels.

Techniques: Expressing, Cell Culture, Western Blot, Staining, Fluorescence, Immunofluorescence