This work was supported by Faculty Research grants from the College of Arts and Sciences Dean's Office at the University of Hartford.

During the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the University of Hartford Animal Institutional Animal Care and Use Committee.
1. Entrainment of Animals
NOTE: Twenty week-old male B6129SF2/J mice are used in the study.
<ol>
	<li>Entrain mice to a 12 h light (standard overhead white light)/12 h dark cycle for 2 weeks prior to the experiment. 
...

<ol>
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Neurosci. 35, 445-462 (2012).</li><li>Balsalobre, A., Damiola, F., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serum+shock+induces+circadian+gene+expression+in+mammalian+tissue+culture+cells.">A serum shock induces circadian gene expression in mammalian tissue culture cells.</a> Cell. 93, 929-937 (1998).</li><li>Yoo, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc. Natl. Acad. Sci. U. S. 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N., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+expression+of+clock+genes+in+mouse+macrophages,+dendritic+cells,+and+B+cells.">Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells.</a> Brain. Behav. Immun. 26, 407-413 (2012).</li><li>Kawai, T., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pattern-recognition+receptors+in+innate+immunity+update+on+Toll-like+receptors.">The role of pattern-recognition receptors in innate immunity update on Toll-like receptors.</a> Nat. Publ. Gr. 11, 373-384 (2010).</li><li>Marpeg&#225;n, L., Bekinschtein, T. A., Costas, M. A., Golombek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+responses+to+endotoxin+treatment+in+mice.">Circadian responses to endotoxin treatment in mice.</a> J. Neuroimmunol. 160, 102-109 (2005).</li><li>Okada, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+LPS+Causes+Transient+Suppression+of+Biological+Clock+Genes+in+Rats.">Injection of LPS Causes Transient Suppression of Biological Clock Genes in Rats.</a> J. Surg. Res. 145, 5-12 (2008).</li><li>Haimovich, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+endotoxin+synchronizes+and+suppresses+clock+gene+expression+in+human+peripheral+blood+leukocytes.">In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes.</a> Crit. Care Med. 38, 751-758 (2010).</li><li>Curtis, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+control+of+innate+immunity+in+macrophages+by+miR-155+targeting+Bmal1.">Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 7231-7236 (2015).</li><li>Silver, A. C., Arjona, A., Walker, W. E., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Circadian+Clock+Controls+Toll-like+Receptor+9-Mediated+Innate+and+Adaptive+Immunity.">The Circadian Clock Controls Toll-like Receptor 9-Mediated Innate and Adaptive Immunity.</a> Immunity. , (2012).</li><li>Gibbs, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+receptor+REV-ERB+&#945;+mediates+circadian+regulation+of+innate+immunity+through+selective+regulation+of+inflammatory+cytokines.">The nuclear receptor REV-ERB &#945; mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines.</a> PNAS. 109, 582-587 (2012).</li><li>Zee, P. C., Attarian, H., Videnovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+abnormalities.">Circadian rhythm abnormalities.</a> Contin. Lifelong Learn. Neurol. 19, 132-147 (2013).</li><li>Bovbjerg, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+disruption+and+cancer:+sleep+and+immune+regulation.">Circadian disruption and cancer: sleep and immune regulation.</a> Brain. Behav. Immun. 17, 48-50 (2003).</li><li>Fu, L., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock:+pacemaker+and+tumour+suppressor.">The circadian clock: pacemaker and tumour suppressor.</a> Nat. Rev. Cancer. 3, 350-361 (2003).</li><li>Germain, A., Kupfer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIRCADIAN+RHYTHM+DISTURBANCES+IN+DEPRESSION.">CIRCADIAN RHYTHM DISTURBANCES IN DEPRESSION.</a> Hum. Psychopharmacol. 23, 571-585 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Mouse+Care+and+Maintenance.">Basic Mouse Care and Maintenance.</a> JoVE. , (2018).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AVMA+Guidelines+for+the+Euthanasia+of+Animals+:+2013+Edition.">AVMA Guidelines for the Euthanasia of Animals : 2013 Edition.</a> AVMA. , (2013).</li><li>Silver, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-associated+molecular+patterns+alter+molecular+clock+gene+expression+in+mouse+splenocytes.">Pathogen-associated molecular patterns alter molecular clock gene expression in mouse splenocytes.</a> PLoS One. , 12-15 (2017).</li><li>Silver, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daily+oscillations+in+expression+and+responsiveness+of+Toll-like+receptors+in+splenic+immune+cells.">Daily oscillations in expression and responsiveness of Toll-like receptors in splenic immune cells.</a> Heliyon. , 00579 (2018).</li></ol>

The author has nothing to disclose.

Within this protocol, a microvolume spectrophotometer can be used to quantify and assess the purity of the RNA being used in determining gene expression. Nucleic acids absorb UV light at 260 nm, proteins typically absorb light at 280 nm, while other potential contaminants used during an RNA extraction procedure (e.g., phenol) are detectable at 230 nm. Therefore, by assessing the absorbance (A) ratio at 260/280 nm (RNA to protein) and 260/230 nm (RNA to non-protein contaminants) the quality of the RNA can be asse...

The master clock in mammals, which orchestrates 24 h oscillations for nearly all aspects of physiology and behavior, is located within the suprachiasmatic nucleus (SCN) of the hypothalamus1,2. In addition to regulating biological processes on an organismal level, the master clock also synchronizes peripheral cellular clocks throughout the body3,4,5. While the molecular clock machinery consists of at least three interlocking transcriptional-translational feedback loops, the core is comprised of the Period (Per1-3), Cryptochrome (Cry1-2), Bmal1, and Clock genes6,7. Besides maintaining the accurate timing of the core molecular clock, some ancillary clock gene products (e.g., Rev-erb&#945; and Dbp) also regulate expression of non-clock genes, i.e., clock controlled genes (CCGs)6,7.
Functional molecular clocks have been described in various immune tissues (e.g., spleen and lymph nodes)8 and cells (e.g., B cells, dendritic cells, macrophages)8,9. These cells detect and respond to pathogen-associated molecular patterns (PAMPs), conserved microbial components, via innate immune recognition receptors such as Toll-like receptors (TLRs)10. To date, 13 functional TLRs have been described, which recognize microbial constituents such as bacterial cell wall components, flagellar protein, and microbial nucleic acids10. The PAMP, lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria recognized by TLR4, has been shown to alter circadian rhythms at the both organismal and molecular levels. For example, in vivo challenge of LPS induced photic-like phase delays as measured by activity in mice11 and led to reduced clock gene expression in the SCN and liver as determined by in situ hybridization and quantitative PCR, respectively, in rats12. After an in vivo challenge with LPS, analysis of human peripheral blood leukocytes13 and subcutaneous adipose tissue14 revealed altered expression of several clock genes as measured via qPCR. Lastly, ex vivo LPS challenges of human macrophages and mouse peritoneal macrophages, also led to altered clock expression as measured by qPCR14.
Here, we describe a protocol to assess the influence of the PAMPs LPS, ODN1826 (synthetic oligonucleotides containing unmethylated CpG motifs), and heat-killed Listeria monocytogenes (HKLM), recognized by TLR4, TLR9, and TLR2, respectively, on molecular clock gene expression in mouse splenocytes. The protocol includes mouse splenectomy, splenocyte isolation and challenge, RNA extraction, cDNA synthesis, and qPCR to assess expression of several clock genes. This protocol allows for the timely acquisition of a large number of immune cells with very little animal or cellular manipulation, which can then be challenged ex vivo with various PAMPs. The molecular clock has been shown to modulate various aspects of the immune response8,15,16, therefore, disruption of the molecular clock would most likely impair the proper time-dependent variation of the immune response. In addition, since disruptions of circadian rhythms can lead to serious pathologies17,18,19,20, it may be of interest for researchers to challenge splenocytes with a wide range of molecules and assess their influence on the clock.

<table border="0" cellpadding="0" cellspacing="0" style="width:796px;" width="795">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Frosted slides</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:137px;">12-550-343</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell strainers</td>
			<td>Fisher</td>
			<td align="right">22363547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipopolysaccharide&#160;</td>
			<td>InvivoGen</td>
			<td>ltrl-eklps</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ODN1826</td>
			<td>InvivoGen</td>
			<td>Tlrl-1826-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HKLM</td>
			<td>InvivoGen</td>
			<td>Tlrl-hklm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>20012-043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104 or 74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well cell culture plate</td>
			<td>Denville</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tubes</td>
			<td>Corning</td>
			<td align="right">352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td align="right">352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher</td>
			<td align="right">4368814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays b-actin</td>
			<td>ThermoFisher</td>
			<td>Mm00607939_s1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Per2</td>
			<td>ThermoFisher</td>
			<td>Mm00478113_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Rev-erba</td>
			<td>ThermoFisher</td>
			<td>Mm00520708_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Bmal1</td>
			<td>ThermoFisher</td>
			<td>Mm00500226_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Dbp</td>
			<td>ThermoFisher</td>
			<td>Mm00497539_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR machine StepOnePlus</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>ThermoFisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Fast 96-well reaction plate (0.1 ml)</td>
			<td>ThermoFisher</td>
			<td align="right">4346907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Statistical Analysis Software</td>
			<td>Prism 7.0a</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

Mice were sacrificed at ZT13, splenocytes were isolated and challenged ex vivo with the PAMPs LPS, ODN1826, or HKLM. After 3 h, RNA was isolated, and qPCR was used to assess relative expression levels of the molecular clock genes Clock, Per2, Dbp, and Rev-erb&#945; compared to unchallenged control cells. After PAMP challenge, Clock expression levels were not significantly different than expression in the control cells (<strong class="x...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

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.

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7.4K Views.  University of Hartford. 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 fo...