Location
Suwanee, GA
Start Date
6-5-2025 1:00 PM
End Date
6-5-2025 4:00 PM
Description
Embryonic hippocampal rat astrocytes were cultured in T75 Poly-D-Lysine flasks for 13 days before trypsinizing. The incubator for cell cultures were maintained at 37 C, 95% humidity, and 5% CO2. The media was exchanged with fresh media every other day for 10 days. This media exchange procedure was altered when it was discovered that exchanging the media with fresh media every day for another four days, increased the number of cells by over 50 percent (Komiskey, unpublished results).
A 12-well plate will be pre-warmed at 37° C. Forceps will be sterilized using 70% ethanol and a flame under the sterile hood. Using sterile forceps, a 22mm poly-D-lysine coated coverslip will be placed into four separate wells of the 12-well plate to allow for cellular adherence. Two 50ml conical tubes with glucose and non-glucose media and cells, will be reserved from glucose deprivation. Three ml of media and cells will be pipetted onto four or more coverslips in the 12 well plate. The prepared plate will be wrapped in tin foil to prevent media evaporation. Media will be changed after two days to allow cells to adhere. Media will be removed from all four wells and washed with 500µ1 of PBS will be added to each well and removed. This wash will be repeated two more times. Final wash will be removed, and 500ul of 4% formaldehyde added to each well, and incubated for 30 minutes at room temperature. Formaldehyde will be removed, followed by a 10-minute incubation in 0.1 % Triton-X for 10 minutes to allow the cell membrane to be more permeable. Following the incubation, Triton-X will be removed, and 500µ1 of 1% BSA added to each well and blocking will be performed for 30 minutes at room temperature. BSA will be removed, and a 1ml of a 1ug/ml dilution of primary antibody in PBS was added to each well. Primary antibody will be incubated for 1 hour at room temperature, after which it will be removed, and the coverslips will be washed 3 times for 3 minutes per wash. Goat anti-rabbit secondary antibody was prepared (1ml per well at lµg/ml dilution), added and incubated for 1 hour at room temperature in the dark. Washes were performed 3x with 500µ1 of IX PBS at 3 minutes per wash DAPI (4',6-diamidino-2-phenylindole) was prepared at a 1: 1000 dilution, 1ml will be added to each well, and allowed to incubate for 10 minutes in the dark at room temperature. Again, three washes were performed with 500µ1 of PBS at 3 minutes per wash. Finally, 500µ1 of PBS will be added to all wells, and the well plate will be Para filmed to prevent evaporation of PBS while placed in 4C refrigerator before imaging. Mitochondria in the astrocytes were also stained using Mitochondrial Membrane assay kit to determine the dose-dependent ability of sulforaphane and/or new compounds as potential inhibitor of mitochondrial damage in astrocytes. Researchers have reported that phosphorylated Tau can change from fibers to liquid droplets and later released.
Embargo Period
5-28-2027
Confocal Microscopy Visualizing Hyperphosphorylated Tau Preparation of Coverslip for Confocal Microscopy
Suwanee, GA
Embryonic hippocampal rat astrocytes were cultured in T75 Poly-D-Lysine flasks for 13 days before trypsinizing. The incubator for cell cultures were maintained at 37 C, 95% humidity, and 5% CO2. The media was exchanged with fresh media every other day for 10 days. This media exchange procedure was altered when it was discovered that exchanging the media with fresh media every day for another four days, increased the number of cells by over 50 percent (Komiskey, unpublished results).
A 12-well plate will be pre-warmed at 37° C. Forceps will be sterilized using 70% ethanol and a flame under the sterile hood. Using sterile forceps, a 22mm poly-D-lysine coated coverslip will be placed into four separate wells of the 12-well plate to allow for cellular adherence. Two 50ml conical tubes with glucose and non-glucose media and cells, will be reserved from glucose deprivation. Three ml of media and cells will be pipetted onto four or more coverslips in the 12 well plate. The prepared plate will be wrapped in tin foil to prevent media evaporation. Media will be changed after two days to allow cells to adhere. Media will be removed from all four wells and washed with 500µ1 of PBS will be added to each well and removed. This wash will be repeated two more times. Final wash will be removed, and 500ul of 4% formaldehyde added to each well, and incubated for 30 minutes at room temperature. Formaldehyde will be removed, followed by a 10-minute incubation in 0.1 % Triton-X for 10 minutes to allow the cell membrane to be more permeable. Following the incubation, Triton-X will be removed, and 500µ1 of 1% BSA added to each well and blocking will be performed for 30 minutes at room temperature. BSA will be removed, and a 1ml of a 1ug/ml dilution of primary antibody in PBS was added to each well. Primary antibody will be incubated for 1 hour at room temperature, after which it will be removed, and the coverslips will be washed 3 times for 3 minutes per wash. Goat anti-rabbit secondary antibody was prepared (1ml per well at lµg/ml dilution), added and incubated for 1 hour at room temperature in the dark. Washes were performed 3x with 500µ1 of IX PBS at 3 minutes per wash DAPI (4',6-diamidino-2-phenylindole) was prepared at a 1: 1000 dilution, 1ml will be added to each well, and allowed to incubate for 10 minutes in the dark at room temperature. Again, three washes were performed with 500µ1 of PBS at 3 minutes per wash. Finally, 500µ1 of PBS will be added to all wells, and the well plate will be Para filmed to prevent evaporation of PBS while placed in 4C refrigerator before imaging. Mitochondria in the astrocytes were also stained using Mitochondrial Membrane assay kit to determine the dose-dependent ability of sulforaphane and/or new compounds as potential inhibitor of mitochondrial damage in astrocytes. Researchers have reported that phosphorylated Tau can change from fibers to liquid droplets and later released.