This method can help answer key questions in the field of host-pathogen interactions such as what are the unique characteristics of the productively infected cells, what host cofactors allow viruses to establish productive infection, and how to target these cells in therapeutic interventions. The main advantage of this technique is that it provides quantitative measures for both protein and mRNA within single cells, and most reagents are commercially available. Demonstrating the cell-sorting procedure will be Matthew Creegan, a senior technician from the flow cytometry core in Doctor Michael Eller's laboratory.
To begin, in a designated pre-PCR molecular biology workstation, prepare the assay mix by combining 96 gene expression assays into an RNase, DNase free tube making sure to add each assay to a final concentration of 180 nanomolar of forward and reverse primers. Add DNA suspension buffer to achieve the appropriate dilution of the assay mix. For each anticipated 96 by 96 chip array, pipette six microliters of each assay into a designated well of a 96 well PCR plate, then seal the plate with an adhesive.
To begin surface staining viable cells, in a tissue culture biosafety cabinet, first prepare the compensation samples and a master mix of fluorescent antibody cocktail as outlined in the text protocol. Thaw the cryopreserved cells in a 37 degree Celsius water bath for two minutes. After this, add between 5 and two milliliters of the cell suspension to a 15 milliliter tube containing 12 milliliters of PBS.
Centrifuge at 500 G for three minutes at 25 degrees Celsius, then aspirate the PBS and resuspend the pellet in three milliliters of fresh PBS. Transfer this mixture to a five milliliter polystyrene tube. Centrifuge once again at 500 G for three minutes at 25 degrees Celsius.
Aspirate the supernatant, leaving approximately 10 microliters residual PBS. Next, resuspend up to 20 million washed cells in 80 microliters of antibody cocktail. Incubate for 20 minutes at 25 degrees Celsius while protected from light.
After this, add three milliliters of PBS. Centrifuge at 500 G for three minutes and aspirate the supernatant. Thoroughly resuspend the cells in 300 to 500 microliters of PBS.
Filter this solution by pipetting it through a 35 micrometer nylon cell-strainer cap. then keep the cells on ice and protected from light until the sort. Back in the pre-PCR workstation, first prepare the RT preAMP reaction mix in a single RNase, DNase free sterile tube as outlined in the text protocol.
Using a multichannel pipette, dispense 10 microliters of this reaction mix into the desired number of 96 well PCR sort collection plates. Seal the plate with adhesive film and then place the plate on a pre-chilled 96 well aluminum block. After this, establish the cell-sorting gating scheme and prepare the flow cytometric cell sorter as outlined in the text protocol.
Enter the appropriate instrument settings to specify the number and subset of cells to be sorted into each well. Remove the adhesive seal, then FACS sort the cells into the prepared 96 well PCR collection plates. Reseal the plate with fresh adhesive film.
Immediately vortex the resealed plate and then centrifuge at 2000 G for one minute at four degrees Celsius. Thermocycle the plate in a PCR machine with a preheated lid at 50 degrees Celsius for 15 minutes followed by 95 degrees Celsius for two minutes. Finally, thermocycle for 18 cycles of 95 degrees Celsius for 15 seconds and 60 degrees Celsius for four minutes, then in a post-PCR workstation, transfer five microliters of cDNA into 20 microliters of DNA suspension buffer in a new 96 well PCR plate.
To begin, prepare the qPCR assay plate by pipetting four microliters of assay-loading reagent in each well. Next, transfer four microliters of each assay from the 2X assay plate to the qPCR plate. Maintain the qPCR assay plate at four degrees Celsius.
Dispense the control line fluids from the priming syringes into the two intake valves of the chip. Remove the protective plastic from beneath the plate. Place the chip on an IFC controller with the notched side at the A1 position, then select and run the prime script.
For each microfluidic chip, mix 50 microliters of the sample loading reagent with 500 microliters of PCR master mix to prepare the real-time reaction mix. Pipette 4.4 microliters of this reaction mix into each well of a new 96 well PCR plate now designated as the sample plate. Next, pipette 3.6 microliters of the previously diluted cDNA into every well of the sample plate.
After this, transfer five microliters from the assay plate to the corresponding well on the notched side of the chip. Transfer five microliters from the sample plate into the corresponding well on the other side of the chip. Insert the loaded chip into the IFC controller and run the load mix script.
Next, transfer the chip to the BioMark platform and set up the instrument as outlined in the text protocol, then save the chip run file in a designated folder. When the chip run is complete, open the real-time PCR analysis software and then select file, open, to open the chiprun. bml file.
In the upper left corner of the software window, locate chip explorer and chip run summary. Under chip run summary, click on detector setup. Under task, click new and select the container type SBS plate and container format SBS 96.
Click on the button with the ellipsis next to mapping, then select M96-Assay-SBS96.dsp. Click on analysis views. In the qPCR tab under task, select baseline correction for linear derivative and CT threshold method for user detectors.
In the CT thresholds tab, check the initialize with auto box and then click the analyze button. In the upper right quadrant of analysis views, click on the second tab, results table. From the dropdown menu, select heat map view and the heat map with the data will appear.
Under the heat map, click threshold and log graph. Adjust the CT thresholds manually for each detector by clicking on assays, represented by columns on the heat map, and dragging the threshold as necessary to intersect the amplification curves in the exponential phase. When done, click analyze.
Next, export the qPCR data as a csv file. Transfer qPCR data from the BioMark computer to your desktop. Import the data into a spreadsheet or statistical analysis software and map the results by sample and assay positions on the chip.
Create a new column and use a conditional formula to organize the cells into groups based on the expression of viral genes. Under analyze, select fit Y by X and plot gene expression versus group. After this, use FlowJo version nine to open FCS files from FACS sort corresponding to a 96 well plate.
With the file name highlighted, select platform, event number gate, and create indexed sort gates. Individual cells will appear displayed by row. Highlight all 96 cells and then select workspace, export, select all compensated floors.
Under data, select FCS file, click export, and select a designated folder. Drag the new FCS files for individual cells into a new FlowJo workspace. Highlight all of the cells and click add statistics, represented by the sigma button, mean, and all fluorescence parameters.
Open the table editor, then highlight all the floors of the first cell and drag them into the table editor window. In this window, click create and view table. After this, copy and output into a statistical software either by copy-pasting or by clicking the save and launch application button.
Merge the single cell FACS data with the qPCR data in JMP by the plate number and well position. Finally, perform graphical and statistical analyses on the combined data. In this study, single cell surface protein quantitation by multi-parameter flow cytometry is integrated with quantitative single cell mRNA expression by highly multiplexed RTqPCR.
Shown here is single cell quantitative viral gene expression from FACS sorted Rhesus macaque CD4 positive T cells. Tat/rev positive N of negative cells express fewer copies of tat/rev RNA than the N of positive cells in dark green which is consistent with an early stage of infection prior to stabilization and a nuclear export of partially spliced viral RNA. A high abundance of unspliced Gag-RNA in tat/rev positive N of positive cells is consistent with late stage productive infection during which abundant genomic RNA is expressed and packaged into budding virions.
This trivariate plot displaying the single cell viral gene, host gene, and host surface protein expression shows that surface CD4 and CD3 protein expression is decreased in tat/rev positive cells shown in green even though CD4 and CD3 transcripts are sustained. Once mastered, this technique can be done in two to three days. After watching this video, you should have a good understanding of how to perform targeted single cell prototranscriptional evaluation to address questions pertaining to differentially expressed genes and proteins in virally infected cells.
