The overall goal of this experiment is to induce B lymphocytes to undergo antibody class-switching and plasma cell differentiation and analyze the modulation as well as epigenetic regulation by the expression of HDAC inhibitor mRNA and microRNA in B-cells. This method can help answer key questions in the B-cell antibody response field such as, epigenetic modulation of immunoglobulin class-switching and plasma cell differentiation. The main advantage of this technique is to provide a simple method to modulate B-cell functions and to define the alteration of microRNAs and mRNAs by epigenetic regulators.
After euthanizing a specific pathogen-free C57BL6J mouse and sterilizing the skin according to the text protocol, use autoclaved forceps and dissecting scissors to cut through the skin just below the ribcage to visualize the spleen on the left side of the abdomen, just below the liver. Gently clamp the spleen and cut off all the connecting tissue before extracting the organ. Then place the spleen into a 1.5 milliliter microcentrifuge tube containing 1, 000 microliters of complete RPMI 1640 medium.
Next, insert a 70 micron cell strainer into a 50 milliliter polypropylene conical centrifuge tube and pour the spleen from the microcentrifuge tube onto the cell strainer. Use the tip of a 15 milliliter polypropylene conical centrifuge tube to gently mesh the spleen through the strainer. Then with 15 to 20 milliliters of complete medium, rinse the strainer.
Spin down the cells at 350 x G for five minutes. Discard the supernatant. Use one milliliter of ACK lysis buffer to re-suspend the cell pellet and incubate for one minute.
Then add four milliliters of ACK and incubate the suspension to remove red blood cells from the spleen suspension for an additional four minutes at room temperature with occasional shaking. Then add 25 milliliters of complete medium to quench the lysis reaction. Spin down the cells at 350 x G for five minutes.
Discard the supernatant and add one milliliters of complete medium to re-suspend the cell pellet. Then using a hemocytometer, count the viable cells. In a sterile five milliliter polystyrene round bottom tube, prepare a 0.25 to two milliliter cell suspension of 1 x 10 to the 8th cells per milliliter in complete medium.
Add normal rat serum to the sample. Next, add 50 microliters per milliliter of isolation cocktail to the tube. Then mix the cells by gently pipetting up and down two to three times and incubate the sample at room temperature for 10 minutes.
Add 75 microliters per milliliter of streptavidin encoated magnetic particles to the tube. Gently pipette up and down two to three times and incubate the sample at room temperature for two and a half minutes. Bring the volume up to 2.5 milliliters using complete RPMI 1640 medium and gently pipette two to three times to mix.
Place the opened tube into the magnet and incubate it at room temperature for two and a half minutes. Then pour off the supernatant containing untouched B-cells into a new 15 milliliter conical tube. Use trypan blue and a hemocytometer to count the cells.
Re-suspend the purified B-cells in complete RPMI 1640 medium at a final concentration of 0.3 x 10 to the 6th cells per milliliter. Then, to the wells of a 48 well plate, add one milliliter of purified B-cells at a 0.3 x 10 to the 6th cells per milliliter three microgram per milliliter of LPS from E.Coli, five nanogram per milliliter of IL4 and either zero or 500 micromolar HDI. Incubate the cells at 37 degree celsius and 5%carbon dioxide for 60 hours for QRT PCR and high throughput mRNA and miRNA sequencing analysis or 96 hours for flow cytometry analysis.
After 96 hours of culture, detach the cells from each well by pipetting them up and down several times. Transfer the cell suspension into a 1.5 milliliter microcentrifuge tube. Then, using a bench top centrifuge, spin down the cells at 350 x G for five minutes.
Then, discard the supernatant. Mix a cocktail of HBSS buffer and the following reagents, 1%BSA, 0.5 nanograms per milliliter of conjugated goat anti-mouse IgM antibody, 0.2 nanograms per milliliter of conjugated rat anti-mouse IgG1 monoclonal antibody, 0.2 nanograms per milliliter of conjugated rat anti-mouse B220 monoclonal antibody, 0.2 nanograms per milliliter of PE-Cy7-conjugated rat anti-mouse CD138 monoclonal antibody and two nanograms per milliliter of 7-AAD. Incubate the cells in the dark at room temperature for 30 minutes.
Then use one milliliter of HBSS with 1%BSA to wash the cells. Use a bench top centrifuge to spin down the cells at 1, 500 x G for five minutes. After discarding the supernatant, use 300 microliters of HBSS with 1%BSA to re-suspend the cells and transfer the cell suspension to a round bottom polystyrene tube.
Cover the tube with foil to avoid light exposure. To carry out QRT PCR analysis of mRNA, include the DNAse 1 treatment step. Then, use a commercial total RNA isolation kit that can recover small RNA to extract RNA from 0.2 to 5 x 10 to the 6th cells, following the manufacturer's instructions.
After synthesizing cDNA and performing QRT PCR on the mRNA, carry out RT PCR for microRNA using a cyber green real time PCR master mix by thawing at room temperature, 2x PCR master mix, 250 nanomolar miRNA specific forward primer and a universal reverse primer. After preparing a 25 microliter reaction mix according to the text protocol, add 22.5 microliter aliquots per well to a 96 well PCR plate. Then, add 2.5 microliters of template cDNA to the individual wells.
Tightly seal the plate film. Then centrifuge the plate at 1, 000 x G at room temperature for one minute. Place the plate in the real time cycler and start the cycling program found in the text protocol.
Using the protocol demonstrated in this video, purified B-cells incubated without PS NIL4 for 96 hours can induce 30 to 40%of CSR to IgG1 and approximately 10%of plasma cell differentiation. After treatment with HDI, the CSR to IgG1 decreased to 10 to 20%while plasma cell differentiation decreased to approximately 2%HDI mediated inhibition of CSR was further confirmed by decreased numbers of post-recombination I mu C gamma 1 and mature VHDJHC gamma 1 transcripts. As measured by QRT PCR, the expression of Aicda which is critical for CSR SHM and Prdm1 and Xbp1, which are important for plasma cell differentiation were shown to be inhibited by VPA.
As measured by mRNA sequencing of B-cells stimulated without PS Nil4, only 0.3%of these highly expressed mRNAs were upregulated by HDI more than two fold and only 0.36%of the highly expressed mRNAs, including Aicda, Prdm1 and Xbp1 were reduced by HDI more than 50%The miRNA sequencing analysis of miRNA profiling in B-cells treated with HDI or Nil, showed that HDIs selectively upregulate Aicda and Prdm1 targeting miRNAs. Once mastered, this technique can be done in four and half to five hours. While attempting this procedure, it is important to remember to keep a sterile work environment.
After watching this video, you should have a good understanding of how to induce B-cells to undergo class-switching and plasma cell differentiation and to modulate these functions by epigenetic regulators.
