human intestinal epithelial t84 cells  (ATCC)

Journal: Frontiers in Immunology

Article Title: Micro- and nano-plastics induce inflammation and cell death in human cells

doi: 10.3389/fimmu.2025.1528502

Figure Lengend Snippet: MNPLs especially PET from commercial sources induce human cell inflammation. (A) MNPLs induce inflammatory IL-1β production in human monocytic THP-1 cells. (B) Endotoxin inhibitor Polymyxin B does not block the inflammatory effects of MNPLs on human THP-1 cells. (C) MNPLs induce inflammatory IL-1β production in human PBMC culture. (D) MNPLs induce inflammatory IL-1β production in human whole blood culture. (E) MNPLs induce inflammatory IL-8 production in human T84 intestinal epithelial cells. The cells or whole blood cultures were treated for three to five days with or without MNPLs from commercial sources. N ≥ 3, Mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 compared to vehicle control.

Article Snippet: Human intestinal epithelial T84 cells and monocytic THP-1 cells were obtained from ATCC (Manassas, VA).

Techniques: Blocking Assay, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacteria from gut microbiota associated with diarrheal infections in children promote virulence of Shiga toxin-producing and enteroaggregative Escherichia coli pathotypes

doi: 10.3389/fcimb.2022.867205

Figure Lengend Snippet: Induction of IL-8 secretion by bacteria used in this study. Intestinal cells in culture (T84 cells) were infected with STEC or EAEC or incubated with the supernatant obtained from the overnight growth of E. albertii (EA SP) or C. werkmanii (CW SP) in M9 medium. As a negative control, cells were incubated with DMEM medium (uninfected) or M9 medium (M9). Three hours post-infection/incubation, the level of IL-8 secretion was evaluated by ELISA. Graphed data are the mean of one representative experiment performed in triplicate, with the error bars indicating standard deviation. * p < 0.05 compared to control condition.

Article Snippet: Human colonic T84 intestinal epithelial cells (CCL-248 ATCC) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium, supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (10 μg/ml), at 37°C in 5% CO 2 .

Techniques: Bacteria, Infection, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacteria from gut microbiota associated with diarrheal infections in children promote virulence of Shiga toxin-producing and enteroaggregative Escherichia coli pathotypes

doi: 10.3389/fcimb.2022.867205

Figure Lengend Snippet: Induction of IL-8 secretion by DEC pathotypes in the presence of EA SP. T84 cells were infected with STEC or EAEC in the presence of the supernatant obtained from the overnight growth of E . albertii (EA SP) in M9 (A) or LB medium (B) . As a negative control, we used uninfected cells incubated with EA SP or with the medium used to prepare the supernatant. Three hours post-infection, the level of IL-8 secretion was evaluated by ELISA. Graphed data are the mean of one representative experiment performed in triplicate, with the error bars indicating standard deviation. * p < 0.05 by ANOVA and multiple comparison analysis.

Article Snippet: Human colonic T84 intestinal epithelial cells (CCL-248 ATCC) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium, supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (10 μg/ml), at 37°C in 5% CO 2 .

Techniques: Infection, Negative Control, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacteria from gut microbiota associated with diarrheal infections in children promote virulence of Shiga toxin-producing and enteroaggregative Escherichia coli pathotypes

doi: 10.3389/fcimb.2022.867205

Figure Lengend Snippet: Induction of IL-8 secretion by DEC pathotypes in the presence of EA SP from clinical strains. T84 cells were infected with STEC (A) or EAEC (B) in the presence of the supernatant obtained from five clinical E . albertii strains (4051-6; 1551-2; 0621-6; 4281-7; 1251-6) and a reference strain (DSM-17582). We used uninfected cells incubated with the different EA SP as a negative control. Three hours post-infection, the level of IL-8 secretion was evaluated by ELISA. Graphed data are the mean of one representative experiment performed in triplicate, with the error bars indicating standard deviation. * p < 0.05 by ANOVA and multiple comparison analysis.

Article Snippet: Human colonic T84 intestinal epithelial cells (CCL-248 ATCC) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium, supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (10 μg/ml), at 37°C in 5% CO 2 .

Techniques: Infection, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacteria from gut microbiota associated with diarrheal infections in children promote virulence of Shiga toxin-producing and enteroaggregative Escherichia coli pathotypes

doi: 10.3389/fcimb.2022.867205

Figure Lengend Snippet: Adherence of STEC and EAEC to intestinal cells in the presence of EA SP.T84 cells were infected with STEC (A) or EAEC (B) in the presence of different concentrations of the supernatant obtained from the overnight growth of E. albertii (EA SP). After 3 h of infection, the number of adherent bacteria was determined by counting colony-forming units (CFU) in LB agar plates. Results are expressed as the means ± the standard errors for one of three experiments done in triplicate. Difference between groups were tested by ANOVA and multiple comparison test.

Article Snippet: Human colonic T84 intestinal epithelial cells (CCL-248 ATCC) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium, supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (10 μg/ml), at 37°C in 5% CO 2 .

Techniques: Infection, Bacteria, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacteria from gut microbiota associated with diarrheal infections in children promote virulence of Shiga toxin-producing and enteroaggregative Escherichia coli pathotypes

doi: 10.3389/fcimb.2022.867205

Figure Lengend Snippet: Induction of IL-8 secretion by STEC 86-24 and STEC AGT210 in the presence of EA SP. T84 cells were infected with STEC wild-type strain or STEC double mutant in Lpf fimbriae (AGT210) in the presence of the supernatant obtained from the overnight growth of E. albertii (EA SP). As a negative control, we used uninfected cells incubated with EA SP or with LB, the medium used to prepare the supernatant. Three hours post-infection, the level of IL-8 secretion was evaluated by ELISA. Graphed data are the mean of one representative experiment performed in triplicate, with the error bars indicating standard deviation. * p < 0.05 by ANOVA and multiple comparison analysis.

Article Snippet: Human colonic T84 intestinal epithelial cells (CCL-248 ATCC) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium, supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (10 μg/ml), at 37°C in 5% CO 2 .

Techniques: Infection, Mutagenesis, Negative Control, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Gut Microbes

Article Title: Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

doi: 10.1080/19490976.2020.1771985

Figure Lengend Snippet: AIEC LF82-infected IECs release exosomes that can inhibit autophagy in recipient cells. Exosomes were purified from the culture supernatant of uninfected T84 cells (Exo-uninfected) or T84 cells infected with the AIEC LF82 strain (Exo-LF82), the E. coli K12 MG1655 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) for 12 h. (a) Western blot analysis of the exosomal markers CD63 and CD9, and the negative marker GRP94 using 30 μg of exosomal protein lysate or T84 protein lysate. (b-e) T84 cells were stimulated for 8 h with Exo-uninfected, Exo-LF82, Exo-K12 or Exo-HS (80 μg of exosome/mL of culture medium) and then uninfected (b, c) or infected with LF82 for 4 h (d, e). Representative western blot analysis (b, d) and quantification of LC3-II/β-actin band intensity (c, e) are shown. Data are representative of three independent experiments and are presented as means ± SEM. Statistical analysis was performed using the one-way ANOVA test followed by a Bonferroni posttest. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Cell culture Human intestinal epithelial T84 cells (ATCC CCL-248 TM ) were maintained in an atmosphere containing 5% CO 2 at 37°C in a Dulbecco’s Modified Eagle Medium/Nutrient F-12 HAM culture medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Dutscher), 1% L-glutamine (Gibco), 1% minimal essential medium vitamins 100X free from L-Glutamine (Dutscher), and 1% antibiotic and antimycotic solution (10,000 U of penicillin, 10 mg of streptomycin, and 0.025 mg of amphotericin B per mL; GE Healthcare HyClone).

Techniques: Infection, Purification, Western Blot, Marker

Journal: Gut Microbes

Article Title: Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

doi: 10.1080/19490976.2020.1771985

Figure Lengend Snippet: MiR-30c and miR-130a levels are increased in the exosomes released from AIEC LF82-infected IECs and in exosome-receiving IECs. (a, b) Exosomes were purified from uninfected T84 cells (Exo-uninfected) or T84 cells infected with the AIEC LF82 strain (Exo-LF82), the E. coli K12 MG1655 strain (Exo-K12) or the E. coli HS strain (Exo-HS) for 12 h. The levels of miR-30c and miR-130a in exosome-donor cells (a) and in the purified exosomes (b) were analyzed by qRT-PCR. (c) T84 cells were stimulated with the purified exosomes (80 μg of exosome/mL of culture medium), and the levels of miR-30c and miR-130a in these cells were analyzed by qRT-PCR. Data are representative of three independent experiments and are presented as means ± SEM. Statistical analysis was performed using the one-way ANOVA test followed by a Bonferroni posttest. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Cell culture Human intestinal epithelial T84 cells (ATCC CCL-248 TM ) were maintained in an atmosphere containing 5% CO 2 at 37°C in a Dulbecco’s Modified Eagle Medium/Nutrient F-12 HAM culture medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Dutscher), 1% L-glutamine (Gibco), 1% minimal essential medium vitamins 100X free from L-Glutamine (Dutscher), and 1% antibiotic and antimycotic solution (10,000 U of penicillin, 10 mg of streptomycin, and 0.025 mg of amphotericin B per mL; GE Healthcare HyClone).

Techniques: Infection, Purification, Quantitative RT-PCR

Journal: Gut Microbes

Article Title: Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

doi: 10.1080/19490976.2020.1771985

Figure Lengend Snippet: MiR-30c and miR-130a transferred from exosomes inhibit ATG5 and ATG16L1 expression in exosome-receiving IECs. Exosomes were purified from uninfected T84 cells (Exo-uninfected) or T84 cells infected with the AIEC LF82 (Exo-LF82), the E. coli K12 MG1655 (Exo-K12) or the HS strain (Exo-HS) for 12 h. (a) T84 cells were transfected with ATG5-3ʹ-UTR-luc or ATG16L1-3ʹ-UTR-luc construct, in which the 3ʹ-UTR of ATG5 or ATG16L1 mRNA was cloned downstream a luciferase-coding sequence in the pMIR-REPORT luciferase vector, or with the empty vector. Sixteen hours after transfection, the cells were incubated with Exo-uninfected, Exo-K12,Exo-HS or Exo-LF82 (80 μg of exosome/mL of culture medium) for 8 h. Luciferase activity was measured and normalized to the protein concentration of cell lysate. (b-d) T84 cells were stimulated with exosomes as in afor 8 h, and ATG5 and ATG16L1 expression at mRNA and protein levels were analyzed by qRT-PCR (b) and western blot (c), respectively. (d) Quantification of band intensity in c. Data are representative of three independent experiments and are presented as means ± SEM. Statistical analysis was performed using the one-way ANOVA test followed by a Bonferroni posttest. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Cell culture Human intestinal epithelial T84 cells (ATCC CCL-248 TM ) were maintained in an atmosphere containing 5% CO 2 at 37°C in a Dulbecco’s Modified Eagle Medium/Nutrient F-12 HAM culture medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Dutscher), 1% L-glutamine (Gibco), 1% minimal essential medium vitamins 100X free from L-Glutamine (Dutscher), and 1% antibiotic and antimycotic solution (10,000 U of penicillin, 10 mg of streptomycin, and 0.025 mg of amphotericin B per mL; GE Healthcare HyClone).

Techniques: Expressing, Purification, Infection, Transfection, Construct, Clone Assay, Luciferase, Sequencing, Plasmid Preparation, Incubation, Activity Assay, Protein Concentration, Quantitative RT-PCR, Western Blot

Journal: Gut Microbes

Article Title: Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

doi: 10.1080/19490976.2020.1771985

Figure Lengend Snippet: Inhibition of miR-30c and miR-130a in AIEC-infected T84 cells abolishes the increase in miR-30c and miR-130a levels in exosomes and in exosome-receiving cells. (a) T84 cells were transfected with vehicle or with 50 nM of anti-miR-negative control (anti-miR-NC) or with a combination of anti-miR-30c and anti-miR-130a. One day post-transfection, the cells were uninfected or infected with the AIEC LF82 or the commensal E. coli HS strain for 12 h. The levels of miR-30c and miR-130a were analyzed by qRT-PCR. (b) Naïve T84 cells were transfected with the ATG5-3ʹ-UTR-luc or ATG16L1-3ʹ-UTR-luc construct, in which the 3ʹ-UTR of ATG5 or ATG16L1 mRNA was cloned downstream a luciferase-coding sequence in the pMIR-REPORT luciferase vector. Sixteen hours after transfection, the cells were stimulated with the exosomes purified from the culture supernatant of the cells in a(designated as Exo-uninfected, Exo-LF82 and Exo-HS) for 8 h (80 μg of exosome/mL of culture medium). Luciferase activity was measured at 1 day post-transfection and was normalized to the protein concentration of cell lysate. (c) Naïve T84 cells were stimulated with the purified exosomes as in b for 8 h, and miR-30c and miR-130a levels in the exosome-receiving cells were analyzed by qRT-PCR. Data are representative of three independent experiments and are presented as means ± SEM. Statistical analysis was performed using the one-way ANOVA test followed by a Bonferroni posttest. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Cell culture Human intestinal epithelial T84 cells (ATCC CCL-248 TM ) were maintained in an atmosphere containing 5% CO 2 at 37°C in a Dulbecco’s Modified Eagle Medium/Nutrient F-12 HAM culture medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Dutscher), 1% L-glutamine (Gibco), 1% minimal essential medium vitamins 100X free from L-Glutamine (Dutscher), and 1% antibiotic and antimycotic solution (10,000 U of penicillin, 10 mg of streptomycin, and 0.025 mg of amphotericin B per mL; GE Healthcare HyClone).

Techniques: Inhibition, Infection, Transfection, Negative Control, Quantitative RT-PCR, Construct, Clone Assay, Luciferase, Sequencing, Plasmid Preparation, Purification, Activity Assay, Protein Concentration

Journal: Gut Microbes

Article Title: Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

doi: 10.1080/19490976.2020.1771985

Figure Lengend Snippet: Inhibition of miR-30c and miR-130a in AIEC-infected T84 cells abolishes the inhibitory effect of exosomes on autophagy-mediated AIEC clearance in recipient cells. T84 cells were transfected with vehicle or with 50 nM of anti-miR-negative control (anti-miR-NC) or a combination of anti-miR-30c and anti-miR-130a. One day post-transfection, the cells were uninfected or infected with the AIEC LF82 or the commensal E. coli HS strain for 12 h, and exosomes were purified from the culture supernatant (designated as Exo-uninfected, Exo-LF82 and Exo-HS, respectively). Naïve T84 cells were stimulated with the purified exosomes for 8 h (80 μg exosome/mL of culture medium). (a) ATG5 and ATG16L1 mRNA expression levels in exosome-receiving cells were analyzed by qRT-PCR. Western blot analysis of ATG5, ATG16L1 and LC3 expression (b) and band intensity quantification (c). (d) Exosome-receiving cells were infected with the AIEC LF82 strain, and the number of intracellular LF82 bacteria was determined using gentamicin protection assay. The results are presented as percentage of CFU of LF82 determined at 24 h versus 4 h post-infection, defined as 100%. Data are representative of three independent experiments and are presented as means ± SEM. Statistical analysis was performed using the one-way ANOVA test followed by a Bonferroni posttest. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Cell culture Human intestinal epithelial T84 cells (ATCC CCL-248 TM ) were maintained in an atmosphere containing 5% CO 2 at 37°C in a Dulbecco’s Modified Eagle Medium/Nutrient F-12 HAM culture medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Dutscher), 1% L-glutamine (Gibco), 1% minimal essential medium vitamins 100X free from L-Glutamine (Dutscher), and 1% antibiotic and antimycotic solution (10,000 U of penicillin, 10 mg of streptomycin, and 0.025 mg of amphotericin B per mL; GE Healthcare HyClone).

Techniques: Inhibition, Infection, Transfection, Negative Control, Purification, Expressing, Quantitative RT-PCR, Western Blot, Bacteria