This method can help answer key questions in the fields of immunology and chronobiology, such as how microbial components alter the molecular clock. The main advantages of this technique are that it is easy to perform, the effects of different microbial components on the molecular clock are assessed, and it is highly reproducible. To begin the protocol, prepare culture medium by adding fetal bovine serum to RPMI 1640 to a final concentration of 10%Prepare 10 milliliters of challenge medium for each of the PAMPS to be tested in 50 milliliter tubes by adding the listed PAMP culture medium.
Spray a previously prepared mouse trunk with 70%ethanol and wipe it with a paper towel. Place the mouse on its back, slightly tilted onto its right side. Next, cut away the fur using dissecting scissors along the mouse's left side, about halfway between the front and back legs.
Using forceps, grab the peritoneum and carefully make an incision without damaging the spleen. Remove the spleen with forceps and place it into a sterile 50 milliliter tube containing approximately 10 milliliters of culture media on ice. Then transfer the spleen with two milliliters of culture medium to a small, sterile Petri dish.
Homogenize the spleen by grinding it between the frosted portion of two sterile frosted slides. Once thoroughly homogenized, pipette the two milliliters of culture medium containing the splenocytes through a 40 micron nylon cell strainer into a 50 milliliter tube. Add eight milliliters of cold culture medium to each of the 50 milliliter tubes containing the splenocytes, for a total volume of 10 milliliters per tube.
Determine the number of cells per milliliter using a hemocytometer. Add approximately one times 10 to the sixth cells per well to six-well culture plates. After adding the cells, add three milliliters of culture medium or three milliliters of challenge medium to the cells.
Then incubate the plates at 37 degrees Celsius and 5%carbon dioxide for three hours. Scrape the cells from the bottom of the well using a 1, 000 microliter pipette tip attached to a P1000 micropipette. With the same pipette tip, transfer the medium containing the cells to a 15 milliliter tube.
Pellet the cells at 167 times g for five minutes at room temperature. Remove the supernatant and wash the cell pellet with five milliliters of PBS. After pelleting the cells a second time at 167 times g for five minutes, remove the supernatant and add 600 microliters of lysis buffer to the cell pellet.
Isolate RNA from splenocytes using the RNA extraction kit according to the manufacturer's instructions and perform the optional on-column DNA digestion. Synthesize cDNA for each of the samples using the reverse transcription kit according to manufacturer's instructions. Use 10 microliters of RNA for each of the samples in a 20 microliter total reaction volume.
Pull mRNA from a few control samples into a 0.5 milliliter tube to prepare the cDNA to be used for the standard curve when performing quantitative PCR. Add 10 microliters of 2X reverse transcriptase master mix. At the completion of the reverse transcription, add 10 microliters of water to the reaction tube, which serves as the starting concentration in the dilution series for the standard curve.
Perform a ten-fold dilution series by adding 45 microliters of water into four 0.5 milliliter tubes designated 10 to the minus one, 10 to the minus two, 10 to the minus three, and 10 to the minus four. Add five microliters of the starting concentration one into the second tube, 10 to the minus one, using a P20 micropipetter. Mix by pipetting up and down several times then transfer five microliters from the 10 to the minus one tube into the tube designated 10 to the minus two tube and mix.
Transfer five microliters using a P20 micropipette from the 10 to the minus two tube into the tube designated 10 to the minus three and mix. Finally, transfer five microliters using a P20 micropipetter from the 10 to the minus three tube into the tube designated 10 to the minus four and mix. Prepare the reaction so that it contains 0.5 microliters of the primer probe assay, five microliters of gene expression master mix, two microliters of water, and 2.5 microliters of cDNA.
Pipette the RNA samples into a 96-well qPCR plate. Incubate primer probe assays for various molecular clock genes and include an assay for an endogenous control, and load it into a qPCR machine to determine the relative quantitation of mRNA levels. Within the experimental setup, select quantitation, relative standard curve and TaqMan chemistry.
Next, change the reaction volume to 10 microliters. Enter the relevant information into the plate layout. qPCR was performed on isolated RNA to assess relative expression levels of the molecular clock genes, clock, Per2, Dbp, and Rev-erb alpha.
After PAMP challenge, clock expression levels were not significantly different compared to control cells. Per2 expression levels were significantly elevated in cells challenged with LPS and ODN1826 when compared to unchallenged controls. LPS was the only PAMP to alter Rev-erb alpha expression as mRNA levels were significantly lower than in the unchallenged controls.
Significantly lower mRNA levels were observed for Dbp after challenge with each of the PAMPs when compared to the controls. Following this procedure, molecules other than PAMPs can be used to assess their influence on the molecular clock in splenocytes.
