The overall goal of the following experiment is to utilize high content assays in cellular disease models to quantify pathways affected in neurodegenerative diseases. To accomplish this, a cellular model of the disease phenotype is input into the automated cell culture system, and cells are incubated until the required cell number is reached. Cells are then split, counted and dispensed into 96 well plates containing lentivirus expressing various SH RNAs.
After six days of cellular differentiation, cells are output from the system for immuno staining and high content imaging. Finally, the images are analyzed using a variety of analysis algorithms to quantify features related to cell health. Neuronal outgrowth and mitochondrial function.
Results are also analyzed by two-way in Novas to identify significant interaction effects. This method can expedite answering key questions as to the function of disease related genes and the pathways within within which they function overall. This will then lead to a better understanding of the pathogenic mechanisms involved in neurodegenerative diseases.
To begin the cell culture process, prepare the automated cell culture system for the input of cell culture plates. Load consumables such as pipette tips, cell culture plates, and assay plates into the system. Also ensure there is sufficient cell culture media PBS and trypsin in the system manually.
Seed two omni tray plates with two times 10 to the six cells per plate of the SH SY five Y neuroblastoma cell line. Maintain cells in optimum media with 10%fetal bovine serum FBS place the plates onto the input shelf in the cell culture robot. Using the graphical user interface or gui, select the A adherent cell culture protocol and adjust a adherent cell line specific parameter files so that the confluence threshold is set at 70%set.
Total ization time to two minutes. Instruct the system to prepare new omni tray plates with a seating cell number of two times 10 to the sixth cells per plate. Instruct the system to prepare for assay plates per omni tray plate with a total of 5, 000 cells per well incubate the cells at 37 degrees Celsius and 5%CO2.
The following steps are involved in this Adherence cell culture protocol. Plates are incubated and imaged until the cells reach the predefined confluence threshold. If cells do not reach the confluence threshold within five readings, the plates are removed from the system.
Upon reaching the user-defined confluence threshold cells are washed, tryps, inized and counted. A predefined number of cells are added to new omni trait plates, and if there are sufficient numbers of cells, a specified number of assay plates are transported to the deck and a defined number of cells dispensed into each well. Finally, assay plates can be directly imaged using the integrated microscope or output from the system for further processing to begin the procedure for virus production.
Inoculate LB medium containing ampicillin with bacterial glycerol stocks containing the HRNA vectors grow overnight on the following day, centrifuge the cells and remove the supernatant extract plasmids using the wizard Magnus cell plasmid purification system. The final E EIT containing plasma DNA is collected in 96 well plates. The S-H-R-N-A lentivirus is produced using the RNAi consortium, a lentiviral production in 96 Well plates, all experiments must be conducted in an ML two or BSL two LA Safety Laboratory and all reagents and consumables used must be decontaminated with 30%chlorine after use.
The procedure for lentivirus production will not be demonstrated in this video, but it takes four days and consists of the following steps. First, the packaging cells are seeded day zero on the following afternoon. Day one, the packaging cells are transfected with three lentivirus plasmids 18 hours post transfection.
Day two, the media is replaced with fresh high BSA or high serum media. 24 hours after the media change. Day three, the virus is harvested and the media is replaced again with fresh high BSA or high serum media.
Finally, 24 hours after the first virus harvest, day four, the virus is harvested again and the packaging cells are discarded to calculate the multiplicity of infection or MOI of the lentivirus transduce, SHSY five Y cells with serial dilutions of the virus incubate cells at 37 degrees Celsius, 72 hours post transduction. Visualize GFP expression under a microscope and determine the percentage of GFP positive cells plate S-H-R-N-A lentivirus onto assay plates with an MOI of three. After plating the SH RNA lentivirus onto the assay plates, load the plates into the automated cell culture system for lentiviral transduction.
After 24 hours, the media on the assay plates will be changed to optimum containing 0.5%FBS and 0.1 micromolar retinoic acid. To begin the differentiation process, continue incubation of the assay plates in the differentiation media for five days to ensure maximal knockdown of target gene expression on day five, add H2O two to half of the assay plates for 24 hours to stimulate translocation of the DJ one protein to the mitochondria on day six. Add MIT tracker to the cells at a final concentration of 200 nano molar per well and incubate at 37 degrees Celsius for 30 minutes.
After this, the plates can be transported to the high content or HC imager for imaging. Alternatively, the plates can be exported from the system for further processing such as immuno staining. For high content imaging, a total of 30 fields per well will be imaged using the 20 x objective lens visualized DJ one with the fitzy filter set mitochondria with the trixy filter set beta three tubulin with the S five filter set and nuclei with the UV filter set.
This is an example of composite images of cells acquired by high content imaging. Figure three untreated cells are shown in panel A and cells treated with H2O two are shown in panel B.DJ one is labeled in green, mitochondria in red and nuclei in blue. To determine the average overlap coefficient between DJ one and the mitochondria, analyze the images using the colocalization bio application.
Define regions of interest. ROI as follows, ROIA nucleus figure four A and E and ROIB mitochondria, figure four B and F exclude ROIA from ROIB. To ensure analysis of only the cytoplasm, define the mitochondria as target region one and DJ one as target region two, figure four C and G.Analyze the images using the com compartmental analysis bio application to determine the average intensity of the MIT tracker signal within the mitochondria.
Figure four B and F to trace the average lengths of the neurites from the beta three tubulin staining. Analyze the images using the neuronal profiling bio application figure four D and H next image plates using the opera lx. Automated confocal reader.
Visualize mitochondria with the 561 nanometer laser and nuclei with UV excitation image. A total of 30 fields per well using the 60 x objective lens with water immersion. Finally analyze the images using the spot edge ridge or SER texture features algorithm.
The SER ridge filter transmits intensity in pixels forming ridge like patterns. The more fragmented the mitochondria, the higher the SER ridge score. In this video, high content or HC assays were used to identify modulators for DJ one A protein whose loss causes autosomal recessive Parkinsonism.
This first representative result illustrates an epistatic interaction with DJ one. The bar graph on the left shows normalized fluorescence units and the images on the right show. SHSY five Y cells labeled red with mito tracker for quantification of cell viability.
It was observed that knockdown of DJ one in cells exposed to toxin resulted in a greater loss of cell viability compared to cells infected with scrambled lentivirus. Knockdown of target gene A had a similar effect to that observed in cells with a DJ one knockdown. However, knockdown of both DJ one and target gene A resulted in a significantly greater loss of cell viability than loss of either gene alone, which suggests an epistatic interaction between DJ one and target gene A.This next example is of a gene regulating DJ one translocation.
The bar graph shows the normalized overlap coefficient between DJ one and the mitochondria, which reflects the translocation of DJ one from the cytoplasm to the mitochondria. Panels A through D are images of SHSY five Y cells labeled in green for DJ one, red for mitochondria and blue for nuclei. When cells are exposed to a toxin DJ one translocates from the cytoplasm to the mitochondria, which is quantified by a higher overlap coefficient between DJ one and the mitochondria.
In cells where target gene B has been silenced less DJ one translocates to the mitochondria when cells are exposed to the toxin, this suggests that target gene B is involved in the transport of DJ one to the mitochondria. An example of a gene involved in neuronal outgrowth is shown here. Normalized neurite lengths are shown in the bar graph and the cells used for quantification of neurite lengths are shown in the images with beta three tubulin in green and nuclei in blue.
The knockdown of target gene C in wild type SHSY five Y cells results in a significant increase in neurite length compared to the cells infected with lentivirus expressing scrambled SH RNA. This effect is lost in cells incubated with toxin. Finally, these results show an example of a gene involved in mitochondrial morphology.
Average mitochondrial SER ridge segmentation values for SHSY five Y cells infected with scrambled HRNA or A-S-H-R-N-A targeting gene D are shown in the graph. Image A and image C are composite images of SHSY five Y cells infected with scrambled HRNA or A-H-R-N-A targeting gene D respectively. Mitochondria are colored in red while nuclei are colored in blue.
Image B and D are visualizations of the SER ridge quantification as observed here. Infection of wild type SHSY five Y cells with HRNA targeting gene D results in an increase in the mitochondrial SER ridge segmentation value when compared to cells infected with scrambled lentivirus. This approach can pave the way for researchers in the field of neurodegeneration to explore and annotate the pathways involved in disease pathogenesis in a timely fashion.
This will then lead to better targets for therapeutic development.
