nih 3t3 fibroblasts  (ATCC)

Journal: Aging and Disease

Article Title: The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9

doi: 10.14336/AD.2024.1715

Figure Lengend Snippet: M6A modifications within total mRNA were significantly enhanced during the aging process . ( A ) Left: representative photographs of 2-month and 20-month-old male C57BL/6 mouse; Right: representative photographs of SA-β-gal staining in kidney tissues from young and old C57-BL/6 mice. ( B ) Left: representative photographs of SA-β-gal staining of P3 and P9 MEF cells; Right: the p16 expression levels in P3 and P9 MEF cells. ( C ) Left: representative photographs of SA-β-gal staining of H 2 O 2 -induced premature senescence of NIH/3T3 cells; Right: the p16 expression levels in H 2 O 2 treated NIH/3T3 cells. ( D ) Poly(A)+ RNA was extracted from the brain and liver tissue of young and old C57-BL/6 mice and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( E ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( F ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( G ) Expression levels of m6A modification associated proteins were measured in P3 and P9 MEF cells, and the quantitative results were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; One sample t test (MEF P9 vs 1, METTL3: p =0.0027, t = 9.176).

Article Snippet: NIH/3T3 fibroblasts were procured from the American Type Culture Collection (ATCC).

Techniques: Staining, Expressing, Dot Blot, Control, Modification

Journal: Carbohydrate polymers

Article Title: Nitric oxide-releasing dextran surface with enhanced albumin affinity mitigates infection and foreign body reaction

doi: 10.1016/j.carbpol.2025.124855

Figure Lengend Snippet: Biocompatibility evaluation of surfaces. (A) Surface adsorbed bovine serum albumin after 90 mins incubation at physiology conditions ( N > 4). (B) Indirect contact cytotoxicity screening of sample groups against BJ human fibroblast cells (24h) showed the viability of cells across all sample groups tested ( N > 5). (C) Percent hemolysis of the film samples (N > 4). 2–5 % hemolytic activity is considered slightly hemolytic, and > 5 % activity is considered hemolytic according to ASTM F756. Error bars represent standard deviation. **** represents statistical significance ( p < 0.0001).

Article Snippet: BJ fibroblast cells (BJ CRL-2522) were derived from stocks previously acquired from the American Type Culture Collection.

Techniques: Incubation, Activity Assay, Standard Deviation

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults reveal reduced cytotoxicity towards senescent fibroblasts. (A) Graphical illustration of the NK cell mediated target cell cytotoxicity assay. Target cells were first stained with calcein acetomethoxymethyl (AM), a vital fluorescent dye. Calcein AM is a non‐fluorescent compound that pass the intact cell membrane into the cytoplasm. Hydrolysis of calcein AM by intracellular esterases in live cells generates calcein, a hydrophilic, intensely fluorescent molecule which reliably stays in the cytoplasm. The stained target cells were next co‐cultured with NK cells isolated from young or old human or mice. NK cells exert their cytotoxicity towards target cells through the release of perforin and granzyme B. Upon lysis of target cells, the calcein dye is released and the loss of the dye is measured as a shift in fluorescence intensity by flow cytometry. Dead cells will appear to the left of the histogram, while alive cells on the right side. The percentage of dead cells can then simply be calculated and presented. (B) Graphical scheme depicts the experimental groups: Co‐cultures of NK cells from young adults with senescent human dermal fibroblasts in the top row and NK cells from old adults with senescent HDF in the bottom row. (C) Histogram (bi‐exponential scale) showing cytotoxicity of NK cells from young and old adults on different senescent HDF. RS, replicative senescent HDF, DIS, doxorubicin induced senescent HDF, IR, ionizing radiation induced senescence, CA, chronologically aged HDF (~75 years). The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (D) The graph depicts the percentage of senescent HDF death ( y ‐axis) by NK cells isolated from young and old human adult. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 5. Two tailed Student's t ‐test was used to assess the significance between young and old groups for each of senescence model. (E) Illustration of the experimental design showing cytotoxic activity of NK cells derived from bone marrow and spleen of young and old mice against aged murine dermal fibroblasts (MDF). (F) Histogram depicting cytotoxicity of NK cells from young and old mice on old MDF. The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (G) The graph depicts the percentage of senescent MDF death ( y ‐axis) by NK cells isolated from young and old mice. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 3. Each mouse NK cell sample used in the cytotoxicity assay was the pool of NK cells isolated from three different mice. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups.

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Cytotoxicity Assay, Staining, Membrane, Cell Culture, Isolation, Lysis, Fluorescence, Flow Cytometry, Two Tailed Test, Activity Assay, Derivative Assay, Comparison

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN restores impairment of conjugation, degranulation and mitochondrial ATP generation in old Natural killer cells. (A) Synapse formation with conjugation of NK cells with the target senescent fibroblasts and the tubulin network pulling the perforin and granzyme B containing vesicles in the direction of the synapse. (B) Fusion of the NK cell derived secretory granules with the presynaptic membrane of NK cells and concomitant exposure of CD107a at the cell membrane and the release of perforin and granzyme B into the synaptic cleft towards the target cell. (C & D) Percentage of NK cell conjugation with senescent HDF when co‐cultured for (C) 60 and (D) 90 min at an effector to target (E:T) cell ratio of 1:1. Data were represented as mean (percentage of cell conjugation) ± SEM, N = 3. (E) Degranulation of NK cells when co‐cultured with senescent HDF for 7 h at an effector to target (E:T) cell ratio of 10:1. Data were represented as mean (mean fluorescence intensity) ± SEM, N = 6. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C, D and E. (F) Seahorse flux analysis showed quantification of ATP generated by NK cells treated with vehicle from young donors and from NK cells treated with either vehicle or CASIN from old donors. The ATP generation either by glycolysis or by oxidative phosphorylation and total ATP was assessed. Data were represented as mean (ATP level) ± SEM, N = 7. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (G) Mitochondrial structure showing the chemical structure of the mitochondrial fluorescent probe JC‐1 that can form J‐aggregates (red) and J‐monomers (green) indicating high and low mitochondrial membrane potential, respectively. (H) Flow cytometry analysis of J‐aggregates (red) and J‐monomers (green) of young NK cells treated with vehicle, old NK cells treated with either vehicle or CASIN. (I) The graph depicts the percentage of J‐aggregates (Q2 population of figure H) of young NK treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (percentage of cell with J‐aggregate) ± SEM, N = 6. (J) Quantification of the ratio of J‐aggregates to J‐monomers from young and old NK treated with vehicle and old NK cells treated with CASIN. Data were represented as mean (ratio) ± SEM, N = 5. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in I and J.

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Conjugation Assay, Derivative Assay, Membrane, Cell Culture, Fluorescence, Comparison, Generated, Phospho-proteomics, Flow Cytometry

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults reveal reduced cytotoxicity towards senescent fibroblasts. (A) Graphical illustration of the NK cell mediated target cell cytotoxicity assay. Target cells were first stained with calcein acetomethoxymethyl (AM), a vital fluorescent dye. Calcein AM is a non‐fluorescent compound that pass the intact cell membrane into the cytoplasm. Hydrolysis of calcein AM by intracellular esterases in live cells generates calcein, a hydrophilic, intensely fluorescent molecule which reliably stays in the cytoplasm. The stained target cells were next co‐cultured with NK cells isolated from young or old human or mice. NK cells exert their cytotoxicity towards target cells through the release of perforin and granzyme B. Upon lysis of target cells, the calcein dye is released and the loss of the dye is measured as a shift in fluorescence intensity by flow cytometry. Dead cells will appear to the left of the histogram, while alive cells on the right side. The percentage of dead cells can then simply be calculated and presented. (B) Graphical scheme depicts the experimental groups: Co‐cultures of NK cells from young adults with senescent human dermal fibroblasts in the top row and NK cells from old adults with senescent HDF in the bottom row. (C) Histogram (bi‐exponential scale) showing cytotoxicity of NK cells from young and old adults on different senescent HDF. RS, replicative senescent HDF, DIS, doxorubicin induced senescent HDF, IR, ionizing radiation induced senescence, CA, chronologically aged HDF (~75 years). The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (D) The graph depicts the percentage of senescent HDF death ( y ‐axis) by NK cells isolated from young and old human adult. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 5. Two tailed Student's t ‐test was used to assess the significance between young and old groups for each of senescence model. (E) Illustration of the experimental design showing cytotoxic activity of NK cells derived from bone marrow and spleen of young and old mice against aged murine dermal fibroblasts (MDF). (F) Histogram depicting cytotoxicity of NK cells from young and old mice on old MDF. The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (G) The graph depicts the percentage of senescent MDF death ( y ‐axis) by NK cells isolated from young and old mice. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 3. Each mouse NK cell sample used in the cytotoxicity assay was the pool of NK cells isolated from three different mice. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups.

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Cytotoxicity Assay, Staining, Membrane, Cell Culture, Isolation, Lysis, Fluorescence, Flow Cytometry, Two Tailed Test, Activity Assay, Derivative Assay, Comparison

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN restores impairment of conjugation, degranulation and mitochondrial ATP generation in old Natural killer cells. (A) Synapse formation with conjugation of NK cells with the target senescent fibroblasts and the tubulin network pulling the perforin and granzyme B containing vesicles in the direction of the synapse. (B) Fusion of the NK cell derived secretory granules with the presynaptic membrane of NK cells and concomitant exposure of CD107a at the cell membrane and the release of perforin and granzyme B into the synaptic cleft towards the target cell. (C & D) Percentage of NK cell conjugation with senescent HDF when co‐cultured for (C) 60 and (D) 90 min at an effector to target (E:T) cell ratio of 1:1. Data were represented as mean (percentage of cell conjugation) ± SEM, N = 3. (E) Degranulation of NK cells when co‐cultured with senescent HDF for 7 h at an effector to target (E:T) cell ratio of 10:1. Data were represented as mean (mean fluorescence intensity) ± SEM, N = 6. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C, D and E. (F) Seahorse flux analysis showed quantification of ATP generated by NK cells treated with vehicle from young donors and from NK cells treated with either vehicle or CASIN from old donors. The ATP generation either by glycolysis or by oxidative phosphorylation and total ATP was assessed. Data were represented as mean (ATP level) ± SEM, N = 7. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (G) Mitochondrial structure showing the chemical structure of the mitochondrial fluorescent probe JC‐1 that can form J‐aggregates (red) and J‐monomers (green) indicating high and low mitochondrial membrane potential, respectively. (H) Flow cytometry analysis of J‐aggregates (red) and J‐monomers (green) of young NK cells treated with vehicle, old NK cells treated with either vehicle or CASIN. (I) The graph depicts the percentage of J‐aggregates (Q2 population of figure H) of young NK treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (percentage of cell with J‐aggregate) ± SEM, N = 6. (J) Quantification of the ratio of J‐aggregates to J‐monomers from young and old NK treated with vehicle and old NK cells treated with CASIN. Data were represented as mean (ratio) ± SEM, N = 5. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in I and J.

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Conjugation Assay, Derivative Assay, Membrane, Cell Culture, Fluorescence, Comparison, Generated, Phospho-proteomics, Flow Cytometry

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay