Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: Visualization of unstained cells under microscope in green light growing on a polycarbonate ( A ) and a polyester ( B ) Transwell membrane.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques: Microscopy, Membrane

Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: Visualization of unstained cells growing on a polyester Transwell membrane. Left, cells attached to collagen and poly-L-lysine-coated plates; right, cells attached to collagen-, fibronectin-, and poly-L-lysine-coated plates. The same plates were observed under green light but without any fluorescence treatment.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques: Membrane, Fluorescence

Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: TEER in astrocyte and pericyte co-cultures seeded in a polyester Transwell insert. Data represent values from triplicate wells on each day for 6 days after seeding. The star indicates the cell density that was considered optimal and carried forward for further experiments. TEER, transepithelial electrical resistance.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques:

Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: TEER in four-cell model containing hBMECs, astrocytes, pericytes, and neurons in a polyester Transwell insert either according to the standard setup or with changes to various conditions: ± Zn; ± serum; ± neurons; or hBMECs only. At each point, triplicates are used for rigor and TEER was measured three times. On day 11 after cell seeding, cells on the Transwell insert membrane were fixed and preserved for confocal microscopy study. TEER, trans-epithelial electrical resistance; Zn, zinc; hBMECs, human brain microvascular endothelial cells.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques: Membrane, Confocal Microscopy

Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: Expression of tight junctions (TJs), ZO-1, and claudin 5 on human brain cells, layered on polyester Transwell insert membrane. Staining was performed for ZO-1 (red), claudin 5 (green), and the nucleus (DAPI; blue). TJs were observed on astrocytes and pericytes growing in medium alone ( A ) or with added zinc sulfate ( B ) and on hBMECs in medium alone ( C ) or with zinc sulfate ( D ). Scale bar inside images represents 50 μm. TJ, tight junction; Z0–1, zona occludens; hBMEC, human brain microvascular endothelial cells; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques: Expressing, Membrane, Staining

Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: Expression of ZO-1 (red) and claudin 5 (green) in monoculture of hBMECs on a Transwell membrane ( A ) and determination of claudin 5 on neuron cell from the non-contact four-cell model with neuronal cell marker in red ( B ) and when the medium for the BBB model was supplemented with Zn ( C ). Scale bars: 20 μm in ( A ) and ( B ), 50 μm in ( C ). Zn, zinc; Z0–1, zona occludens; hBMECs, human brain microvascular endothelial cells; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques: Expressing, Membrane, Marker

Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: Cell identification by detecting s100β on astrocytes and CD146 on pericytes and hBMECs as respective cell markers. Astrocytes, pericytes, and hBMECs on a Transwell membrane, immunostained with a cocktail of s100β antibody (red), an antibody against CD146 (green); the nucleus was stained with DAPI (blue). The basolateral surface of the Transwell membrane shows astrocyte and pericyte ( A ), and the apical part of the same Transwell membrane shows hBMECs only ( B ). Scale bars: 50 μm. Abbreviations: hBMECs, human brain microvascular endothelial cells; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques: Membrane, Staining

Journal: Current protocols

Article Title: Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells

doi: 10.1002/cpz1.1067

Figure Lengend Snippet: Average fluorescence intensity analysis of claudin 5 and drug penetration for different sets of BBB representation. ( A ) Cell surface pixel intensity for claudin 5 measured using Fiji ImageJ-win64 software for image analysis. ( B ) Evaluation of tight junction integrity and the penetration of HIV-ART drug DTG in the four-cell mode. The color-coded bar pairs show drug distribution in the apical and bottom layers of the indicated in vitro models and control (membrane only) from a Transwell membrane plate. Medium from the apical and basal layer of the Transwell membrane was collected after 48 hr of drug treatment. Data represent the mean concentrations from three replicates. MFI, mean fluorescence intensity; AP, astrocytes and pericytes; DTG, dolutegravir; hBMECs, human brain microvascular endothelial cells; Zn, zinc.

Article Snippet: 70% (v/v) ethanol Endothelial CM (see recipe) Transwell inserts bearing BBB models (Basic Protocol ) Paraformaldehyde (Thermo Scientific, cat. no. J19943-K2) Dulbecco’s phosphate-buffered saline (DPBS; Gibco, cat. no. 14190144) Goat serum (Abcam, cat. no. ab7481) Tween-20 (Sigma, cat. no. P2287–100ML) Bovine serum albumin (BSA) Wash buffer (see recipe) Appropriately conjugated (labeled) antibodies for claudin 5 (Invitrogen, cat. no. 362588) and ZO-1 (Invitrogen, cat. no. MA3–39100 A647) Appropriately conjugated (labeled) antibodies for cell markers: anti-S1OOβ for astrocytes (Abcam, cat. no. ab196175), anti-CD146 (Abcam, cat. no. ab196448 for pericytes and hBMECs), and anti-NeuN antibody for neuron (Abcam, ab190565) DAPI-(4′,6-diamidino-2-phenylindole) mounting dye (ProLong Diamond Antifade Mounting medium with DAPI, Invitrogen cat. no. P3692) Dolutegravir (DTG; Adooq Bioscience) DTG solution without DTG (see recipe) EVOM2 for TEER measurement (World Precision Instruments [WPI]) Scalpel Scissors Beakers Glass slides Glass coverslips Forceps Measuring cylinders Magnetic stirrer Magnetic beads Vortex Confocal microscope (Zeiss LSM800 microscope) Fiji ImageJ software Liquid chromatography-tandem mass spectrometer (LC/MS/MS)

Techniques: Fluorescence, Software, In Vitro, Control, Membrane