Transfection:Article Title: Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy
Article Snippet: .. HeLa cells; can be obtained from American Type Culture Collection (ATCC) DMEM (Hyclone #SH3002201) FBS (Fisher Scientific #03600511) 0.05% Trypsin–EDTA 1× (Life Technologies #25300054) Phosphate-buffered saline (PBS) 1× (Hyclone #SH30256.01) Mounting medium with DAPI (Vector Laboratories #H1200; Life Technologies Prolong Gold # ) Microcentrifuge tubes (Dot Scientific #010) Coverslips, No. 1.5 (Dot Scientific #MC22-15) Poly- l -lysine (Sigma #P1399) Formaldehyde (Sigma #X100) Triton X-100 (Sigma #F1635) Slides (Fisher Scientific #125442) 10 cm dishes (Falcon #353003) 6-well dishes (Falcon #353046) Primary antibodies against CXCR4 (BD Transduction Laboratories #BDB551852); LAMP2 (Developmental Studies Hybridoma Bank #H4B4), EEA1 (BD Transduction Laboratories #BDB610456), β-arrestin1 (kindly provided by Jeffrey L. Benovic, Thomas Jefferson University, Philadelphia, PA) Secondary antibodies (Molecular Probes); goat anti-rat IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 649) #A21094; goat anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 488 conjugate) # siRNA (Dharmacon) against Luciferase (P-002099), β-arrestin1 (siGenome SMARTpool #M-011971) Lipofectamine 3000 Transfection Reagent (Life Technologies #L3000008) Opti-MEM (Life Technologies #31985062) Hemacytometer (Fisher Scientific #0267154) Tissue paper (Fisher Scientific #06666A) Tweezers Lens paper Whatman filter paper Parafilm Confocal microscope equipped with 60× objective ..
Luciferase:Article Title: Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy
Article Snippet: .. HeLa cells; can be obtained from American Type Culture Collection (ATCC) DMEM (Hyclone #SH3002201) FBS (Fisher Scientific #03600511) 0.05% Trypsin–EDTA 1× (Life Technologies #25300054) Phosphate-buffered saline (PBS) 1× (Hyclone #SH30256.01) Mounting medium with DAPI (Vector Laboratories #H1200; Life Technologies Prolong Gold # ) Microcentrifuge tubes (Dot Scientific #010) Coverslips, No. 1.5 (Dot Scientific #MC22-15) Poly- l -lysine (Sigma #P1399) Formaldehyde (Sigma #X100) Triton X-100 (Sigma #F1635) Slides (Fisher Scientific #125442) 10 cm dishes (Falcon #353003) 6-well dishes (Falcon #353046) Primary antibodies against CXCR4 (BD Transduction Laboratories #BDB551852); LAMP2 (Developmental Studies Hybridoma Bank #H4B4), EEA1 (BD Transduction Laboratories #BDB610456), β-arrestin1 (kindly provided by Jeffrey L. Benovic, Thomas Jefferson University, Philadelphia, PA) Secondary antibodies (Molecular Probes); goat anti-rat IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 649) #A21094; goat anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 488 conjugate) # siRNA (Dharmacon) against Luciferase (P-002099), β-arrestin1 (siGenome SMARTpool #M-011971) Lipofectamine 3000 Transfection Reagent (Life Technologies #L3000008) Opti-MEM (Life Technologies #31985062) Hemacytometer (Fisher Scientific #0267154) Tissue paper (Fisher Scientific #06666A) Tweezers Lens paper Whatman filter paper Parafilm Confocal microscope equipped with 60× objective ..
Protease Inhibitor:Article Title: Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition
Article Snippet: .. Rabbit anti ZO-1 (Cat # 617300), mouse anti ZO-1 (Cat # 339100), 0.25% Trypsin (1X), Opti-MEM (1X)/reduced serum medium, Dulbecco's modified Eagle’s medium (DMEM; with high glucose, HEPES, no phenol red (1X)), NuPAGE Novex® 10% Bis-Tris protein gels, NuPAGE® MOPS SDS Running Buffer, NuPAGE® Transfer Buffer, HyClone Dulbecco's phosphate buffered saline (PBS, without calcium, magnesium, or phenol red), TRIzol® Reagent, SuperScript® IV First-Strand Synthesis System, Halt® Protease Inhibitor Cocktail (100X), Pierce™ ECL Western Blotting substrate and rhodamine phalloidin were purchased from Thermo Fisher Scientific (Carlsbad, CA). .. Anti-MMP-9 antibody (cat # ab-3) was obtained from EMD Millipore (Billerica, MA).
Article Title: Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma
Article Snippet: .. Tissue dissociation and cell purification Tissues were transported in RPMI 1640 (Gibco, Cat. no. 11875–093) with 1 mM protease inhibitor (Solarbio, Cat. no. P6730) on ice to preserve viability, washed 2–3 times with phosphate buffered saline (PBS; Hyclone, Cat. no. SH30256.01), then minced on ice. .. We used dissociation enzyme cocktail 1 mg/ml Type VIII Collagenase (Sigma-Aldrich, Cat. no. C2139), 2 mg/ml Dispase II (Sigma-Aldrich, Cat. no.4942078001), 1 mg/ml Trypsin Inhibitor (Sigma-Aldrich, Cat. no. T6522) and 1 unit/ml DNase I (NEB, Cat. no. M0303S) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Gibco, Cat. no. 16000–044) to digest the tissues.
Microscopy:Article Title: Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy
Article Snippet: .. HeLa cells; can be obtained from American Type Culture Collection (ATCC) DMEM (Hyclone #SH3002201) FBS (Fisher Scientific #03600511) 0.05% Trypsin–EDTA 1× (Life Technologies #25300054) Phosphate-buffered saline (PBS) 1× (Hyclone #SH30256.01) Mounting medium with DAPI (Vector Laboratories #H1200; Life Technologies Prolong Gold # ) Microcentrifuge tubes (Dot Scientific #010) Coverslips, No. 1.5 (Dot Scientific #MC22-15) Poly- l -lysine (Sigma #P1399) Formaldehyde (Sigma #X100) Triton X-100 (Sigma #F1635) Slides (Fisher Scientific #125442) 10 cm dishes (Falcon #353003) 6-well dishes (Falcon #353046) Primary antibodies against CXCR4 (BD Transduction Laboratories #BDB551852); LAMP2 (Developmental Studies Hybridoma Bank #H4B4), EEA1 (BD Transduction Laboratories #BDB610456), β-arrestin1 (kindly provided by Jeffrey L. Benovic, Thomas Jefferson University, Philadelphia, PA) Secondary antibodies (Molecular Probes); goat anti-rat IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 649) #A21094; goat anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 488 conjugate) # siRNA (Dharmacon) against Luciferase (P-002099), β-arrestin1 (siGenome SMARTpool #M-011971) Lipofectamine 3000 Transfection Reagent (Life Technologies #L3000008) Opti-MEM (Life Technologies #31985062) Hemacytometer (Fisher Scientific #0267154) Tissue paper (Fisher Scientific #06666A) Tweezers Lens paper Whatman filter paper Parafilm Confocal microscope equipped with 60× objective ..
Purification:Article Title: Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma
Article Snippet: .. Tissue dissociation and cell purification Tissues were transported in RPMI 1640 (Gibco, Cat. no. 11875–093) with 1 mM protease inhibitor (Solarbio, Cat. no. P6730) on ice to preserve viability, washed 2–3 times with phosphate buffered saline (PBS; Hyclone, Cat. no. SH30256.01), then minced on ice. .. We used dissociation enzyme cocktail 1 mg/ml Type VIII Collagenase (Sigma-Aldrich, Cat. no. C2139), 2 mg/ml Dispase II (Sigma-Aldrich, Cat. no.4942078001), 1 mg/ml Trypsin Inhibitor (Sigma-Aldrich, Cat. no. T6522) and 1 unit/ml DNase I (NEB, Cat. no. M0303S) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Gibco, Cat. no. 16000–044) to digest the tissues.
Concentration Assay:Article Title: A Synthetic Tetramer of Galectin-1 and Galectin-3 Amplifies Pro-apoptotic Signaling by Integrating the Activity of Both Galectins
Article Snippet: .. For all apoptosis experiments, 100 μL of cells were aliquoted at 200,000 cells into round-bottom 12 × 75 mm culture test tubes (14-956-3D, ThermoFisher) and incubated with 100 μL of sterile 1x PBS (Hyclone™ SH30256) alone (i.e., untreated), G1, G3, G1 + G3, or G1/G3 Zipper in sterile 1x PBS (final galectin concentration depending on assay) in the presence or absence of 100 μM caspase-8 inhibitor Z-IETD-FMK (FMK007, R & D Systems), caspase-3/7 inhibitor I (218826, MilliporeSigma), ERK inhibitor U0126 (662005, MilliporeSigma), or JNK inhibitor II SP600125 (420119, MilliporeSigma) for 4 or 24 h at 37°C, 5% CO2 . .. Note, inhibitors were dissolved in American Chemical Society grade dimethyl sulfoxide (DMSO) and an equivalent amount of DMSO (1 μL or 0.5% final concentration) was added to all groups not receiving inhibitors as vehicle control.
Article Title: Host Immune Response Triggered by Graphene Quantum-Dot-Mediated Photodynamic Therapy for Oral Squamous Cell Carcinoma
Article Snippet: .. For observation of the cellular uptake of GQD-PEG, SCC 9 and human oral keratinocytes (HOK) were seeded in confocal Petri dishes (15 mm) at a density of 104 cells/mL and cultured for 24 h. Cells were then incubated with GQD-PEG or pristine GQD at a concentration of 50 μg/mL for 6 h and washed 3 times with phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA). .. For the tracking of intracellular distribution, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed by confocal laser scanning microscopy (CLSM; Zeiss, Jena, Germany).
Incubation:Article Title: A Synthetic Tetramer of Galectin-1 and Galectin-3 Amplifies Pro-apoptotic Signaling by Integrating the Activity of Both Galectins
Article Snippet: .. For all apoptosis experiments, 100 μL of cells were aliquoted at 200,000 cells into round-bottom 12 × 75 mm culture test tubes (14-956-3D, ThermoFisher) and incubated with 100 μL of sterile 1x PBS (Hyclone™ SH30256) alone (i.e., untreated), G1, G3, G1 + G3, or G1/G3 Zipper in sterile 1x PBS (final galectin concentration depending on assay) in the presence or absence of 100 μM caspase-8 inhibitor Z-IETD-FMK (FMK007, R & D Systems), caspase-3/7 inhibitor I (218826, MilliporeSigma), ERK inhibitor U0126 (662005, MilliporeSigma), or JNK inhibitor II SP600125 (420119, MilliporeSigma) for 4 or 24 h at 37°C, 5% CO2 . .. Note, inhibitors were dissolved in American Chemical Society grade dimethyl sulfoxide (DMSO) and an equivalent amount of DMSO (1 μL or 0.5% final concentration) was added to all groups not receiving inhibitors as vehicle control.
Article Title: Host Immune Response Triggered by Graphene Quantum-Dot-Mediated Photodynamic Therapy for Oral Squamous Cell Carcinoma
Article Snippet: .. For observation of the cellular uptake of GQD-PEG, SCC 9 and human oral keratinocytes (HOK) were seeded in confocal Petri dishes (15 mm) at a density of 104 cells/mL and cultured for 24 h. Cells were then incubated with GQD-PEG or pristine GQD at a concentration of 50 μg/mL for 6 h and washed 3 times with phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA). .. For the tracking of intracellular distribution, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed by confocal laser scanning microscopy (CLSM; Zeiss, Jena, Germany).
Article Title: Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia
Article Snippet: .. BV2 cells were seeded in the transwell chambers (Cat. No. MCRP24H48, Millipore) at a density of 2 × 104 cells/chamber or 1 × 105 cells/plate for 6 h before treatment with lipopolysaccharides (LPS, 1ug/mL) (Cat. No. L4516, Sigma) for 24 h. For further incubation with mNPC-exos, BV2 chambers were washed with warmed PBS (Cat. No. SH30256.01, Hyclone), moved into 661 W wells, added fresh mNPC-exos in the upper chamber and co-cultured till 48 h for further assay. .. Human NPC were isolated from human embryonic retina which was provided by the embryonic tissue bank of the Department of Obstetrics in Southwest Hospital [ ].
Cell Culture:Article Title: Host Immune Response Triggered by Graphene Quantum-Dot-Mediated Photodynamic Therapy for Oral Squamous Cell Carcinoma
Article Snippet: .. For observation of the cellular uptake of GQD-PEG, SCC 9 and human oral keratinocytes (HOK) were seeded in confocal Petri dishes (15 mm) at a density of 104 cells/mL and cultured for 24 h. Cells were then incubated with GQD-PEG or pristine GQD at a concentration of 50 μg/mL for 6 h and washed 3 times with phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA). .. For the tracking of intracellular distribution, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed by confocal laser scanning microscopy (CLSM; Zeiss, Jena, Germany).
Modification:Article Title: Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition
Article Snippet: .. Rabbit anti ZO-1 (Cat # 617300), mouse anti ZO-1 (Cat # 339100), 0.25% Trypsin (1X), Opti-MEM (1X)/reduced serum medium, Dulbecco's modified Eagle’s medium (DMEM; with high glucose, HEPES, no phenol red (1X)), NuPAGE Novex® 10% Bis-Tris protein gels, NuPAGE® MOPS SDS Running Buffer, NuPAGE® Transfer Buffer, HyClone Dulbecco's phosphate buffered saline (PBS, without calcium, magnesium, or phenol red), TRIzol® Reagent, SuperScript® IV First-Strand Synthesis System, Halt® Protease Inhibitor Cocktail (100X), Pierce™ ECL Western Blotting substrate and rhodamine phalloidin were purchased from Thermo Fisher Scientific (Carlsbad, CA). .. Anti-MMP-9 antibody (cat # ab-3) was obtained from EMD Millipore (Billerica, MA).
Western Blot:Article Title: Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition
Article Snippet: .. Rabbit anti ZO-1 (Cat # 617300), mouse anti ZO-1 (Cat # 339100), 0.25% Trypsin (1X), Opti-MEM (1X)/reduced serum medium, Dulbecco's modified Eagle’s medium (DMEM; with high glucose, HEPES, no phenol red (1X)), NuPAGE Novex® 10% Bis-Tris protein gels, NuPAGE® MOPS SDS Running Buffer, NuPAGE® Transfer Buffer, HyClone Dulbecco's phosphate buffered saline (PBS, without calcium, magnesium, or phenol red), TRIzol® Reagent, SuperScript® IV First-Strand Synthesis System, Halt® Protease Inhibitor Cocktail (100X), Pierce™ ECL Western Blotting substrate and rhodamine phalloidin were purchased from Thermo Fisher Scientific (Carlsbad, CA). .. Anti-MMP-9 antibody (cat # ab-3) was obtained from EMD Millipore (Billerica, MA).
Immunofluorescence:Article Title: In vitro model of the glial scar
Article Snippet: .. 2.5 Immunofluorescence StainingThe embedded astrocytes were fixed with 4% paraformaldehyde for an hour and washed with 1× PBS (phosphate-buffered saline, HyClone, SH30256.01) 6 times at 10 min intervals. .. Then, the method of freezing slice was used to obtain 40 µm thickness slices for the immunofluorescence staining of the embedded cells.
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