We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.
The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.
Blood-brain barrier (BBB) is the protective barrier limiting the influx of plasma components into the brain tissue and consists of brain endothelial cells along with supporting cells such as pericytes. The major role of BBB is to serve as a barrier that seals the space between peripheral blood and central nervous system (CNS) to maintain hemostasis of the neural microenvironment1,2. The brain capillary endothelial cells effectively seal the paracellular pathway via formation of intercellular tight junctions (TJs)1. This protective barrier allows glucose and selected nutrients to enter the brain while it prevents the majority of ions, toxic substances and drugs from passing through this tight barrier. Apart from its protective role, the natural barrier function of the BBB poses a severe challenge in the development of drug delivery systems targeting the CNS.
In vitro cell culture models of the BBB are helpful tools to study its biology and to understand the effects of drug treatment on TJ barrier integrity. We used the human cerebral microvascular endothelial cell line (hCMEC/D3) as an in vitro model since it is an accepted model of human brain endothelium3 and recapitulates many functions of the human BBB. hCMEC/D3is one of the most commonly used cell lines for modeling the BBB in vitro4,5,6,7,8,9. Despite its comparatively low values of transendothelial electrical resistance (TEER), a measure of barrier tightness, this cell line retains most of the morphological and functional properties of brain endothelial cells, even as a monoculture in the absence of cocultured glial cells6,7. The hCMEC/D3 cell line expresses multiple BBB markers including active transporters and receptors until approximately passage 35 without undergoing dedifferentiation to unstable phenotypes6,7,9,10,11. The most striking characteristic of hCMEC/D3 cell line as an in vitro BBB model is its ability to form TJs5,9,11,12. It should be noted that although stem cell-derived BBB models showed higher permeability in many studies compared with hCMEC/D3 cell line and they do express some BBB markers, they are yet to evolve as the most common BBB cell model13. Importantly, stem-cell derived BBB models remain to be characterized with respect to maximum passage numbers that allow the cells to maintain stable BBB phenotypes14.
Three primary methods are commonly used to determine the TJ barrier integrity, including the measurement of TEER, measurement of apparent permeability coefficient (Papp) of small hydrophilic tracer molecules such as sucrose, inulin, Lucifer Yellow, etc. and immunostaining of known molecular markers of TJs such as claudin-5, ZO-1, occludin, etc.5. TEER is a relatively simple and quantitative method that measures the electrical resistance across the cell monolayers cultured on a porous membrane substrate5. However, TEER values can be influenced by experimental variables such as composition of the culture medium and the type of measurement instrument. A likely combination of these factors leads to a broad distribution of TEER values ranging from 2 to 1150 Ω cm2 in the hCMEC/D3 cell line cultured for 2-21 days13. Immunostaining is a visual method to determine the presence of TJ proteins by labelling the targeted protein using antibodies. However, immunostaining involves a series of experimental steps, including the need to fix/permeabilize cells that may result in experimental artifacts and the fluorescent signals may fade over time. The above factors may lead to subjective errors affecting data quality.
The primary focus of this work is to present a LY-based apparent permeability assay determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells. Although other advanced in vitro BBB systems, such as co-culture systems, microfluidic systems, are physiologically more relevant mimics with significantly improved barrier function15,16,17, the hCMEC/D3 transwell setup is a simple and reliable model to estimate the kinetics of TJ formation and rapidly screen the effect of different drug formulations on barrier function. In general, Papp values are consistent for various hydrophilic solutes in hCMEC/D3 monolayers. For example, the reported Papp values for various low molecular mass solutes (such as sucrose, mannitol, LY, etc.) in different in vitro BBB models are in the order of 10-4 cm/min5,18,19,20. In our experimental setup, the brain endothelial cells are seeded on a collagen-coated microporous membrane for cell attachment and monolayer formation to mimic the in vivo barrier. The LY added in the apical side is expected to traverse the intercellular tight junctions and accumulate in the basolateral side. Greater concentrations of LY in the basolateral side indicate an immature, not-fully functional barrier while lower concentrations reflect restricted transport due to the presence of functional TJs resulting in a mature barrier.
LY is a hydrophilic dye with distinct excitation/emission peaks and avoids the need to radiolabel tracer molecules such as sucrose, mannitol or inulin. Thus, the fluorescence values of LY can be used to directly calculate its paracellular permeability across the BBB monolayers. Also, compared to many commercially available dyes used in biomedical fields that suffer from small Stokes shifts such as fluorescein21, the Stokes shift of LY is about 108 nm with sufficient spectral separation, thus allowing LY fluorescence data as a robust readout to determine paracellular permeability. We used Western blotting as an orthogonal technique to demonstrate changes in expression of the tight junction marker protein, ZO-1, over culture time. ZO-1 expression detected via Western blotting is used to supplement the LY Papp data and in combination, these data suggest that the observed changes in LY Papp values is reflective of the formation of a monolayer with gradual increase in expression of the tight junction marker, ZO-1.
As pointed out earlier, the central focus of this work is to demonstrate a LY assay as a simple technique to monitor the formation of a confluent monolayer with functional tight junctions. However, to demonstrate an additional utility of the developed assay, we measured the LY Papp in DNA nanoparticle-transfected hCMEC/D3 monolayers. Nucleic acids can be condensed into polyelectrolyte nanoparticles with a diameter of 100-200 nm via electrostatic interaction between the positively charged groups of polymers and the negatively charged phosphate groups of nucleic acids22,23. We refer to these complexes as DNA nanoparticles (DNA NPs) in our work. While our intention is to transfect cells and express the desired protein, we must ensure that the barrier properties of the hCMEC/D3 monolayers are not compromised. Our data suggests that a standard 4 h luciferase gene transfection regime does not measurably change the LY permeability demonstrating the utility of the LY Papp assay to determine changes in TJ barrier integrity.
1.General hCMEC/D3 cell culture
2. Cell plating
3. Kinetics of cell growth.
4. Lucifer yellow apparent permeability (LY P app ) assay
5. Calcium depletion
6. Transfection
7. Luminescent ATP assay
8. Western blotting for measurement of tight junction protein ZO-1
First, we determined the effect of culturing time on LY permeability to determine the apparent kinetics of TJ formation. The mean LY Papp values from day 1 to 10-post seeding are shown in Figure 2a. On day 1, the mean Papp was 4.25 x 10-4 cm/min and slightly dropped to 3.32 x 10-4 cm/min on day 2. The mean Papp value slightly increased to 3.93 x 10-4 cm/min on day 3 and fluctuated with no significant changes until day 6. The Papp values significantly decreased to 2.36 x 10-4 cm/min on day 7 compared to day 1 (P < 0.05) probably suggesting that the barrier became tighter. The Papp values stabilized in the range between 2.14 x 10-4 and 2.36 x 10-4 cm/min from day 7 to day 10, which implied the barrier formation was complete and functional resulting in decreased LY paracellular transport. We calculated the percentage (%) LY recovered on each day to be ca. 80%, a value that is considered optimal to reliably calculate the Papp value27 (Figure 2b). Recovery% is an important index in LY assay. For example, if cells metabolize most LY, LY is immobilized in cells or sticks to the cell membrane or LY degrades during incubation, it would be inaccurate to interpret that observed low LY signal in basolateral compartment indicates a tight barrier. Thus, recovery% gives more confidence that we did not lose significant amount of LY owing to one or more of the above possibilities and allows to confidently estimate LY Papp values. As noted earlier in the introduction section, greater concentrations of LY in the basolateral side indicates an incomplete barrier while lower concentrations reflect restricted transport, suggesting a mature, complete barrier due to the presence of functional TJs. We also present additional evidence using an orthogonal technique, Western blotting detection of ZO-1 protein (Figure 4), to confirm that the observed changes in LY Papp correlates with the formation of tight junctions.
Since the transwell insert setup does not allow to directly track changes in cell density, we determined changes in cell density using a standard Trypan blue exclusion assay. We therefore determined the changes in cell density on a transparent tissue culture plate that readily allowed us to monitor the cell growth kinetics. The increase in cell density from 5.5 ± 1.0 x 104 cells/cm2 to 1.9 ± 0.2 x 105 cells/cm2 from day 1 to 10-post seeding (Figure 3) was linear with a regression coefficient of 0.94. These data also suggest that the observed changes in LY Papp (Figure 2a) are a result of the formation of a confluent monolayer over the 10-day period. We observed the cells under an inverted light microscope on each day and visually documented a gradual increase in cell number and monolayer formation.
We used Western blotting to detect changes in the expression of the tight junction protein ZO-1 over time (Figure 4). The changes in ZO-1 expression is used to orthogonally supplement the LY Papp data and to ensure that the observed changes in LY Papp indicates the formation of a tight barrier. ZO-1 band intensities were analyzed by densitometry and normalized relative to the expression of a housekeeping gene, GAPDH. The two bands in Figure 3a represent the two ZO-1 isoforms (ZO-1α+ and ZO-1α–)28. Densitometry analysis revealed that the pixel value of ZO-1 increased from day 3-7 post-seeding, suggesting that the TJ protein ZO-1 formed continually from day 3-7. After calcium depletion treatment on day 7 post-seeding, the band of ZO-1 was almost undetectable, which indicates that ZO-1 was unable to form in the absence of calcium ions. Moreover, the pixel value of ZO-1 markedly decreased at day 10 post-seeding. The signal intensities of GAPDH from day 3-10 appeared were comparable, except on day 7 when the cells were treated with calcium-free medium. A possible reason for the lower GAPDH expression in calcium-treated cells may be due to the lesser total protein (28.9 µg total protein), again, likely due to calcium depletion. Overall, the band densitometry analysis revealed a gradual increase in ZO-1 expression (relative to GAPDH) until day 7 and a decrease in expression on day 10 when cells cultured in complete growth medium. The analysis also revealed a lower expression of ZO-1 (relative to GAPDH) on day 7 in cells treated with calcium-free medium.
While the focus of this work is to present the LY Papp assay as a method to determine the kinetics of a monolayer formation, to demonstrate an additional utility of the developed assay, we determined whether DNA NP transfection influenced the TJ barrier integrity by measuring LY Papp through hCMEC/D3 cells 4 h post-transfection (Figure 5). Our DNA NPs containing Poloxamer P84 mediate high levels of gene expression in the hard-to-transfect hCMEC/D3 cell line (Figure 6a). Specifically, we wanted to determine if the hydrophobic domains of Poloxamer P84 in our DNA NPs may perturb TJ integrity in transfected cells. The %LY recovered in each treatment group was ca. 92%, suggesting that the calculated Papp values are reliable (Figure 5b). We noted that the transfection procedure using various formulations did not affect the LY Papp, relative to non-transfected cells exposed to LY alone. Extracellular calcium is a critical component for the maintenance of cell-cell junctions in various cell types29,30,31, including the brain microvessel endothelial cells. Thus, maintaining cells in calcium-free medium (CFM) leads to the disruption of TJs32,33. Therefore, we used cells treated with CFM for 24 h as a positive control.
Our data shows that the LY Papp for cells incubated with CFM was 2-fold higher compared to control cells incubated with the regular growth medium. This 100% increase in Papp suggests that the cells had lost their TJs because of the loss of calcium ions needed for its formation. Notably, the actual value (5.14 x 10-4 cm/min) was slightly higher than the average Papp value from day 1-6 post seeding (Figure 2) when the TJs were not yet fully formed. Albeit not significant, cells transfected with DNA NP containing 0.01-0.03% Poloxamer P84 showed a tiny increase in LY Papp values compared to the untreated cells maintained in regular culture medium. This observation suggested that DNA NP + P84 transfection had no significant effect on TJ barrier integrity. Overall, the LY Papp values in the transfected cells approximately averaged to 2.5×10-4 cm/min and this value corresponded to the average value noted during day 7-10 post-seeding (Figure 2) when the LY Papp was the least, suggesting the presence of functional TJs that effectively restricted LY paracellular transport.
We present additional transfection data to show that the lack of changes in LY Papp (Figure 5) is not an inconsequential observation. Despite the high levels of transfection observed in the DNA NPs+P84 group, we noted no changes in LY Papp suggesting that our formulations do not perturb the TJ barrier. The relatively low transfection efficiency in the naked DNA-treated cells is typical because the anionic nature of plasmid DNA (due to phosphate groups in its backbone) and its hydrophilic nature limit cellular uptake. DNA condensed in DNA NP mediated a 50-fold increase in transfection compared to naked pDNA (Figure 6). Addition of 0.01% P84 to the DNA NP resulted in an 18-fold increase compared to DNA NP-alone (P< 0.01). Increasing the P84 concentration to 0.03 wt.% resulted in a 30-fold increase compared to DNA NP-alone (P< 0.001). These results are noteworthy given that brain endothelial cells are a hard-to-transfect cell type.
We measured the cell viability of cells transfected under different conditions to confirm that the transfection procedure does not cause cell stress. Adenosine triphosphate (ATP) is the energy currency of life and reflects cellular metabolic function. We used a luciferase-based ATP assay where the luminescence values are directly proportional to ATP levels. Possimo et al. reported that the luminescence ATP assay was a robust measure of metabolic cell viability34. The cell viability of hCMEC/D3 cells transfected by the various formulations was comparable to untreated cells (Figure 5b), suggesting that the ATP levels were similar as well. Therefore, DNA NPs containing Poloxamer P84 (0.01% to 0.03% w/w) are safe gene delivery formulations.
Figure 1. Experimental setup for LY Papp study. 24-well plate setup containing transwell inserts (adapted to show DNA nanoparticle transfection as an example, data in Figure 5). Each column was treated with the indicated sample for 4 h. Control indicates hCMEC/D3 cells treated with complete growth medium while calcium depletion 24 h indicates that the cells were incubated with calcium free medium prior to LY exposure. The right template depicts LY fluorescence intensity measurement in a black 96-well plate. Please click here to view a larger version of this figure.
Figure 2. Apparent kinetics of TJ barrier formation determined using the LY Papp assay. (a)LY Papp through hCMEC/D3 monolayers cultured on transwell inserts were measured everyday post-seeding cells. (b) %LY recovered in each treatment group. Data represents average ± SD of two independent experiments (n=3/experiment). Statistical comparisons were made using unpaired t-test (*P< 0.05, N.S. not significant). Please click here to view a larger version of this figure.
Figure 3. Kinetics of cell growth determined using a Trypan blue exclusion assay. hCMEC/D3 cells were seeded in a 24-well plate at a cell density of 50,000 cells/cm2. On each day of the experiment, cells were dissociated and mixed with an equal volume of 0.4% Trypan blue before counting viable cells on a hemacytometer. Data represents average ± SD of three independent measurements. Please click here to view a larger version of this figure.
Figure 4. Apparent kinetics of ZO-1 expression detected using western blotting. (a) The hCMEC/D3 cells were seeded in a 12-well plate at a cell density of 50,000 cells/cm2. On each day of the experiment (day 3, day 5, day 7 and day 10-post seeding), cells were lysed by 400 µL of 1x RIPA buffer containing 3 µg/mL aprotinin. Cell lysates containing 40 µg of total protein were loaded on a 4-7.5% SDS-polyacrylamide gel. (b) Band densitometry analysis allowed normalizing expression of ZO-1 protein to GAPDH. Please click here to view a larger version of this figure.
Figure 5. Effects of DNA NP transfection on TJ barrier tightness measured using the LY Papp assay. (a) hCMEC/D3 cells were cultured on transwell inserts for 7 days, transfected with PEG-DET containing gWIZLuc plasmid DNA with/without Pluronic P84 for 4 h and then replaced with pre-warmed transport buffer containing 50 µM LY for 1 h. Control represents the hCMEC/D3 cells incubated with growth medium for 4 h followed by LY exposure (n=4, *P<0.05, N.S. not significant). (b) %LY recovered in each treatment group, values presented are average ± SD (n = 4). Please click here to view a larger version of this figure.
Figure 6. DNA NPs mediate high levels of transgene expression in hCMEC/D3 monolayers. (a) hCMEC/D3 cells were cultured 7 days and transfected with PEG-DET containing gWIZLuc plasmid DNA with/without Pluronic P84 (N/P 10, DNA dose per well: 0.5 µg) for 4 h, transfection mixture was removed and cells were cultured for 24 h in complete growth medium prior to measuring gene expression. Levels of luciferase gene expression was expressed as relative light units (RLU) nornalized to total cellular protein content. Data presents average ± SD of three independent experiments. Statistical comparisons were made using unpaired t-test (** P< 0.01, *** P< 0.001). (b) DNA NPs are safe transfection formulations in hCMEC/D3 monolayers. Effects of DNA DNA NP transfection on cell viability was evaluated using a luminescent ATP assay. hCMEC/D3 cells were transfected with the indicated samples for 4 h following which the ATP assay was conducted by following manufacturer's protocol. Percent (%) cell viability was calculated as follows: (luminescence of transfected cells/luminescence of control, untreated cells)x100. Data represents average ± SD of two independent experiments (n=3/experiment). Please click here to view a larger version of this figure.
Experimental setup | Sample name | Volume of 1mg/mL plasmid DNA (µL) | Volume of 10 mM NaAc buffer, pH 5(µL) | Volume of 5 mg/mL polymer (µL) | Volume of 10% w/w. P84 (µL) | Volume of growth medium (µL) |
Tissue culture insertsa | Control, untreated cells | 0 | 0 | 0 | 0 | 58.3 |
Naked DNA | 0.157 | 8.143 | 50 | |||
DNA NP | 7.843 | 0.3 | ||||
DNA NP + 0.01%P84 | 7.6681 | 0.1749 | ||||
DNA NP + 0.03%P84 | 7.26 | 0.0583 | ||||
48-well plate | Control, untreated cells | 0 | 0 | 0 | 0 | 175 |
Naked DNA | 0.5 | 24.5 | 150 | |||
DNA NP | 23.56 | 0.94 | ||||
DNA NP + 0.01%P84 | 23.385 | 0.175 | ||||
DNA NP + 0.03%P84 | 23.035 | 0.525 | ||||
96-well plate | Control, untreated cells | 0 | 0 | 0 | 0 | 68.1 |
Naked DNA | 0.195 | 58.4 | ||||
DNA NP | 9.35 | 0.37 | ||||
DNA NP + 0.01%P84 | 8.9307 | 0.0681 | ||||
DNA NP + 0.03%P84 | 9.0669 | 0.2043 |
Table 1. The NP formulations used for obtaining the data reported.
A key role of the BBB is to prevent the exchange of non-essential ions and toxic substances between the systemic circulation and the brain to maintain hemostasis of neural microenvironment. One of the characteristic features of the BBB is the ability of the capillary endothelial cells to form tight junctions (TJs) that effectively seal the paracellular route of transport. We demonstrated a LY Papp assay as a quantitative method to determine the apparent kinetics of TJ barrier formation in cultured hCMEC/D3 monolayers. ZO-1 expression detected via Western blotting orthogonally validated the data from the LY Papp studies as discussed in detail in the following paragraph. As an additional utility of the developed assay, we further demonstrated that DNA NP transfection did not measurably change the LY Papp indicating the suitability of this assay to determine changes in TJ barrier characteristics in an experimental setup.
Western blot data revealed a clear increase in ZO-1 expression on days 3, 5, 7-post seeding and a slight reduction on day 10-post seeding (Figure 4a). From Figure 1, it can be seen that the LY Papp decreased from day 1-7 post-seeding suggesting the formation of TJs. After calcium depletion treatment, the LY Papp showed a marked increase (Figure 2a) while the ZO-1 expression showed marked reduction (Figure 4a). Extracellular calcium is a critical component for the maintenance of cell-cell junctions in various cell types29,30,31, including the brain microvessel endothelial cells. Lack of calcium in the growth medium disrupted and dissociated the TJs33. As expected, the Papp value of calcium-depleted cells was significantly higher than the untreated cells and close to the Papp value of the blank inserts containing no cells (Figure 2a). The LY revealed a steady plateau in Papp values from day 7-10 post-seeding (Figure 2a) suggesting that the barrier had fully formed by day 7 that resulted in restricted LY transport to the basolateral side. The Western blot data showed a slight decrease in ZO-1 expression on day 10 compared to day 7-post seeding. This difference in observation is likely due to the contribution of other extracellular proteins, apart from ZO-1, in maintaining barrier tightness. In summary, we have successfully used data from two orthogonal techniques to determine the kinetics of tight junction barrier formation in a human cell model of the BBB. While the LY Papp assay measured the functionality of the TJ barrier, the Western blot data traced the formation of the TJs using ZO-1 as a marker protein.
As an additional utility of the developed assay, we confirmed that DNA NP transfection in hCMEC/D3 monolayers does not affect the integrity of TJs (Figure 5). Untreated cells incubated with growth medium was used as the negative control and cells pre-incubated with calcium free medium for 24 h was used as a positive control. Cells in which the TJs were disrupted as a result of the calcium depletion showed a 210% increase in LY Papp compared to the untreated cells. Our data suggests that DNA NP transfection either in the presence or absence of P84 does not affect the TJ integrity. It should be noted that the lack of LY Papp changes in transfected cells is not an inconsequential observation. In fact, our DNA NPs mediate significant levels of gene expression in the hard-to-transfect hCMEC/D3 monolayers9,35,36,37. DNA NPs containing 0.01 or 0.03 wt. % P84 increased luciferase gene expression by ca. 18-and 30-fold, respectively, compared to DNA NPs alone (Figure 6a). We also demonstrated that our NP treatments do not adversely affect the cellular ATP levels, used here as an indicator of functional cell viability (Figure 6b). Our results underscore the expanded utility of this LY Papp assay to determine the TJ barrier integrity in an experimental transfection setup.
One critical step in the execution of the LY assay is to keep the same amount of LY in apical side and equal volume of transport buffer in the basolateral side across the various time points in the entire experiment. If different amounts of LY or unequal volumes of transport buffer were used in wells, the calculation of Papp would be unreliable, resulting in artificially large standard deviations. Another critical aspect is to limit the light exposure to minimize the decay of LY fluorescence intensity. Also, the liquid needs to be removed completely before adding exact same volume of LY solution and transport buffer into apical side and basolateral side, respectively. This ensures that the fluorescence readout can be reliably used for Papp calculation. Another critical step in this experiment is to immediately measure the LY fluorescence of the basolateral samples and avoid the need to freeze the samples after collection. Subjecting the LY-containing samples to freeze-thaw cycles results in a larger intra-group variation of the fluorescence signal. One limitation of using the transwell setup is that the live cells are difficult to be visualized by conventional microscopy or imaged by confocal microscopy. Moreover, it does not easy translate into a setup that allows co-culturing different cell types unless mixed culturing of say, glial and endothelial cells is a possibility. Nevertheless, the LY assay has the following advantages: LY is a dye with distinct excitation/emission peaks and a relatively large Stokes shift compared to dyes like sodium fluorescein, which allows for a robust measurement of the probe crossing the BBB and avoids the need for additional radiolabel as in the case of tracers such as sucrose or mannitol. High recovery %s of LY provides reliable data to confidently calculate Papp values.
Based on our findings, we conclude that the LY Papp method is a simple and robust assay to quantify the paracellular permeability across hCMEC/D3 cell monolayers. By using LY Papp method, we optimized the culture time for the formation of intact barrier, and we demonstrated an additional utility of the developed assay by determining TJ integrity in DNA NP-transfected cell monolayers.
The authors have nothing to disclose.
The authors are thankful for the financial support from the 2017 New Investigator Award from the American Association of Pharmacy, a Hunkele Dreaded Disease award from Duquesne University and the School of Pharmacy start-up funds for the Manickam laboratory. We would like to thank the Leak laboratory (Duquesne University) for western blotting assistance and allowing use of their Odyssey 16-bit imager. We would also like to include a special note of appreciation for Kandarp Dave (Manickam laboratory) for help with western blotting.
hCMEC/D3 cell line | Cedarlane Laboratories | 102114.3C-P25 | human cerebral microvascular endothelial cell line |
gWizLuc | Aldevron | 5000-5001 | Plasmid DNA encoding luciferase gene |
lucifer yellow CH dilithium salt | Invitrogen | 155267 | |
Transwell inserts with polyethylene terephthalate (PET) track-etched membranes | Falcon | 353095 | |
Tissue culture flask | Olympus Plastics | 25-207 | |
24-well Flat Bottom | Olympus Plastics | 25-107 | |
Black 96-Well Immuno Plates | Thermo Scientific | 437111 | |
S-MEM 1X | Gibco | 1951695 | Spinner-minimum essential medium (S-MEM) |
EBM-2 | Clonetics | CC-3156 | Endothelial cell basal medium-2(EBM-2) |
phosphate-buffered saline 1X | HyClone | SH3025601 | |
Collagen Type I | Discovery Labware, Inc. | 354236 | |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23227 | |
Cell Culture Lysis 5X Reagent | Promega | E1531 | |
Beetle Luciferin, Potassium Salt | Promega | E1601 | |
SpectraMax i3 | Molecular Devices | Fluorescence Plate Reader | |
Trypan Blue Solution, 0.4% | Gibco | 15250061 | |
ZO-1 Polyclonal Antibody | ThermoFisher | 61-7300 | |
anti-GAPDH antibody | abcam | ab8245 | |
Alexa Fluor680-conjugated AffiniPure Donkey Anti-Mouse LgG(H+L) | Jackson ImmunoResearch Inc | 128817 | |
12-well, Flat Bottom | Olympus Plastics | 25-106 | |
RIPA buffer (5X) | Alfa Aesar | J62524 | |
Aprotinin | Fisher BioReagents | BP2503-10 | |
Odyssey CLx imager | LI-COR Biosciences | for scanning western blot membranes |