Current studies typically use permeability tests and associated protein expression to verify the accuracy of TER detection. The BBB model of monolayer cells in vitro is less effective for drug screening and the use of more cells makes its more difficult to construct the model. The protocol of this study is relatively simple and the non-invasive procedure allows the cell samples to be used in other experiments after TER detection.
The detection of TER to assess the permeability of cell models can accelerate the screening of drugs for treatment and prevention of the central nervous system to increase the success rate of new drug development. In the future, we will detect the expression of tight junction protein in upper compartment cells of To verify that, TER test results can effectively be present in the integrity of BBB barrier function. To begin, prepare DMEM cell culture medium containing FBS, penicillin, and streptomycin.
Also, prepare 100 mM cobalt chloride stock solution by adding 1.30 mg of cobalt chloride to 100 L DMSO. Next, seed 1 mL of bEnd. 3 cells in a 100 mL culture dish containing DMEM medium and culture at 37 C in a humidified atmosphere of 5%carbon dioxide.
Change the medium every two to three days and subculture the cells two times a week. After bEnd. 3 cells grow to 80%confluence, digest the cells with 0.25%trypsin for 30 seconds.
Count the cells using a cell counter and make a suspension of density 7 x 10^4 cells per mL. Then seed 100 L of this cell suspension in a 96-well plate for cell adhesion. Then wash the cells with PBS and add 100 L of culture medium containing cobalt chloride.
Culture the cells for 24 hours. After incubation, remove the medium and wash with PBS. Next, add 100 L of CCK-8 solution.
Incubate the plates at 37 C for one hour. Then measure the absorbance at 450 nm using a microplate reader. Calculate the cell viability induced by cobalt chloride according to this formula.
Select the concentration with a significant difference in cell viability reduction compared with the control group. The viability of bEnd. 3 cells treated with different concentrations of cobalt chloride was analyzed by CCK-8 assay.
The results indicated that 300 M of cobalt chloride showed significant in vitro cytotoxicity. To begin, rinse the upper chamber of 24-well plates with PBS. Mix the bEnd.
3 cells with DMEM medium using a vortex mixer to make a suspension. Then seed 200 L of bEnd. 3 cell suspension on the PET membrane in the upper chamber of a 24-well plate.
Add 1, 200 L of complete medium to the lower chamber of the plate to stabilize the osmotic pressure of the upper and lower chambers. Change the medium by slowly removing the old medium from one side with a negative pressure pipette and adding a new medium along the wall during fluid exchange. Before measuring TEER, place the resistor 5%sodium hypochlorite solution, 75%ethanol, and double distilled water on an ultra-clean table.
Turn on the UV irradiation for 30 minutes to eliminate residual bacteria and pathogens. Place the electrodes in 5%sodium hypochlorite solution with slow shaking for three to five seconds. Then immerse in 75%ethanol for 15 minutes.
Finally, transfer to PBS or double-distilled water until use. Next turn on the switch at the back of the cell resistor meter. Click select plate, and select 24-well plate.
According to the operation, select the appropriate detection sequence. Insert a kilo ohm resistor into the right plug to calibrate the instrument. If the calibration result is 1, 000 5 ohms, consider the instrument accurate.
If not, click on mode units on the main interface to select ohms and then click calibrate. Then remove the kilo ohm resistor and replace it with the measuring electrode using a connecting wire. Place the electrode vertically into the 24-well plate containing only medium and click blank handling on the instrument.
The background value of the plate resistance without seeded cells should be about 134.4 ohm. Next, insert the two electrodes into the upper and lower chambers of the seeded plate, such that the cell layer is between them. Record the resistance value by gently stepping on the pedal.
Make sure that the electrode does not touch the cells in the upper chamber and the bottom of the lower chamber. Obtain TEER values by multiplying electrical resistance values with the bottom area of the upper chamber as shown in the equation. Draw a line plot of TEER vs time in days.
When the resistance value does not increase with time, the cells have formed a barrier. The resistance values plotted vs time for bEnd. 3 cells showed that the cell TEER values began to stabilize on the fifth day.
Begin by culturing the bEnd. 3 cells, then divide the wells into the control group and cobalt chloride group. Add 200 L of culture medium into the upper chamber of the control group and culture medium containing 300 M cobalt chloride into the cobalt chloride group.
Incubate the plates at 37 C and detect the resistance values of the at 12 and 24 hours of incubation using TEER method. Use commercial software to map the trend of TEER values of the cell barrier. Use statistical analysis software to analyze the difference in resistance values between cobalt chloride treated cells and control cells.
Addition of 300 M cobalt chloride resulted in a significant decrease in TEER values compared to the control at both the 12 hour and 24 hour time points. After 24 hours, the TEER value of 300 M cobalt chloride group reduced the TEER value of 300 M cobalt chloride group reduced compared to the control group, indicating breakdown of the cell barrier that was induced by hypoxic conditions due to cobalt chloride.
