The overall goal of this procedure is to efficiently transfect primary human T cells with small RNA reagents using next generation electroporation. This method can help answer key questions in T cell biology, such as how specific gene products regulate T cell differentiation and immune function. The main advantage of this technique is that it is a highly efficient and reliable method to transfect primary human T cells with sRNAs and other synthetic small RNA reagents.
This method can provide insight into the biology of human Th17 cells, but it can also be applied to other systems, such as other lymphocytes from humans or mice. On day zero, coat six well tissue culture plates with 1.5 milliliters per well of antihuman CD3 and antihuman CD28 in PBS with calcium and magnesium for at least two hours at 37 degrees Celsius. Once the cord-blood mononuclear cells, or CBMCs, have been isolated and washed, perform CD4 positive T cell isolation by negative selection using a commercial human CD4 positive T cell kit.
Add isolation buffer and re-suspend the mononuclear cells. Transfer the cells to a new five milliliter tube, then add 100 microliters of fetal bovine serum, or FBS, and 100 microliters of the antibody mix per tube, then incubate each tube at four degrees Celsius for 20 minutes on an orbital shaker to mix well. After the incubation, add three to four milliliters of isolation buffer to wash the cells.
Centrifuge the cells at 300 times g for eight minutes at four degrees Celsius to pellet the cells. Carefully aspirate the supernatant. Transfer magnetic beads into a new tube and add an equal volume of isolation buffer to wash the beads.
Use a one milliliter micropipet to mix the beads well and then place the tube in the magnet for at least one minute. Carefully aspirate the supernatant and re-suspend the magnetic beads in the same volume initially transferred prior to the wash. Add 500 microliters of the isolation buffer to each tube and re-suspend the cells, then add 500 microliters of pre-washed magnetic beads.
Incubate the cells with the magnetic beads for 15 minutes on an orbital shaker at room temperature to mix well. After incubation, using a one milliliter micropipet, thoroughly pipet the cells at least 10 times, then add three to four milliliters of isolation buffer and place each tube in the magnet for two minutes. Carefully transfer the negatively selected CD4 positive T cells that are in the supernatant to a new tube.
Once CD4 positive T cell isolation is completed, keep the cells on ice and count the cells with a hemocytometer. Wash the antibody coated plates two times with PBS, then add 1.5 milliliters of two X mix of Th17 polarizing media to each well. Next, centrifuge the human CD4 positive T cells to pellet the cells.
Carefully aspirate the supernatant and discard it, then re-suspend the cells in serum free base media and plate the cells so that the final volume is three milliliters per well and polarizing cytokines are now at a one X final concentration, then place the plates in 5%carbon dioxide in a 37 degrees Celsius incubator for two days. On day two, coat 48 well tissue culture plates with 250 microliters per well of antihuman CD3 and antihuman CD28 in PBS with calcium and magnesium for at least two hours at 37 degrees Celsius. To prepare small RNAs for transfection, aliquot one microliter of a five micromolar stock solution of siRNA into a 1.5 milliliter microcentrifuge tube for each transfection.
Keep all of the tubes on ice. Next, wash the antibody coated plates two times with PBS, then add 500 microliters of one X Th17 polarizing media to each well. After the culture plates and transfection reagents are prepared, re-suspend the cells with a one milliliter micropipet, pipetting gently but ensuring that all the cells are detached from the bottom of the wells.
Pool the cells into a conical tube and centrifuge the tubes at 500 times g for five minutes at four degrees Celsius. Carefully aspirate the supernatant and then re-suspend the cells in at least one milliliter of PBS to wash. Count the live Th17 cells with a hemocytometer, using trypan blue exclusion to assess viability, then pellet the cells in a microcentrifuge tube.
Carefully aspirate the supernatant and re-suspend the cells using the provided re-suspension buffer from the kit. Keep the cells at room temperature. Add nine microliters of cells to one microliter of small RNA in each microcentrifuge tube.
Pipet once to mix the cells and small RNAs for transfection, then load the cells into the provided pipet electrode tip. To ensure optimal transfection, avoid introducing air bubbles into the pipet electrode tip prior to transfection. We add 9.5 microliters of cell suspension to ensure there is enough mixture to prevent creating bubbles in the electroporation pipet.
Fill the provided cuvette with three milliliters of room temperature electrolytic buffer. Place the cuvette inside the neon pipet station and then place the pipet into position inside the cuvette. Immediately electroporate each 10 microliter mix of cells and small RNA.
After the electroporation is complete, directly add the cell mixture to 500 microliters of one X Th17 polarizing media in prepared wells of a culture plate. Finally, place the plates in a 5%carbon dioxide, 37 degrees Celsius incubator for two more days. Work carefully but quickly to prevent cell death.
If there are many samples to electroporate, the cells can be collected and transfected in batches. This contour plot shows that T cells cultured under Th17 polarizing conditions expressed the chemokine receptor, CCR6, and the transcription factor, ROR gamma T, which were not expressed when T cells were cultured under non-polarizing conditions. T cells cultured under Th17 polarizing conditions also produced IL-17A upon re-stimulation, but not under ThN conditions.
Th17 differentiation still occurs after transfection with small RNAs, but in some experiments, there is an observed reduction in the frequency of IL-17A production. This demonstrates the importance of having a chemistry matched small RNA control for comparison within each experiment. Viability is maintained after transfection with small RNAs and is typically greater than 70%Transfection of human Th17 cells on day two with an siRNA pool targeting the gene, PTPRC, which encodes CD45, strongly reduced the expression of CD45 compared to a chemistry matched control siRNA.
The unimodal population shift in CD45 expression, seen here, indicates near 100%transfection efficiency. Reducing ROR gamma T expression by RNAi had a functional effect, as these cells exhibited reduced IL-17A production compared to cells transfected with a control siRNA. After watching this video, you should have a good understanding of how to transfect in vitro polarized human Th17 cells with small RNAs using next generation electroporation.
