Name: nf-&#954;b-dependent luciferase activation and quantification of gene expression in salmonella infected tissue culture cells
Uploaded: 2020-01-12T15:00:00
Description: Watch this Scientific Journal Video about NF-ÎºB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells at JoVE.com

Research in the Keestra-Gounder lab is supported by grants from the NIAID of the NIH under Award Number R21AI122092 and from the American Diabetes Association under Award Number 1-18-JDF-035.

1. Cell Passaging and Seeding
<ol>
	<li>Maintain HeLa 57A cells in a 75 cm2 flask containing 10 mL of Dulbecco&#39;s modified Eagle&#39;s media (DMEM) supplemented with 5% heat inactivated fetal bovine serum (FBS) at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Aspirate cell culture media and wash with 1 mL of 0.05% trypsin-EDTA solution. Aspirate trypsin and replace with an additional 1 mL. Place the flask in a 37 &#176;C incubator for 4-5 min to allow cells to detach from flask.</li>
	<li>Add 9 m...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB:+a+stress-regulated+switch+for+cell+survival.">NF-kappaB: a stress-regulated switch for cell survival.</a> Antioxidants &amp; Redox Signaling. 8 (3-4), 478-486 (2006).</li><li>Lawrence, T. <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+factor+NF-kappaB+pathway+in+inflammation.">The nuclear factor NF-kappaB pathway in inflammation.</a> Cold Spring Harbor Perspectives in Biology. 1 (6), a001651 (2009).</li><li>Hargett, D., Rice, S., Bachenheimer, S. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+cross-linking+method+for+identification+of+NF-kappaB+gene+network+by+chromatin+immunoprecipitation.">Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.</a> Biotechniques. 39 (5), 715-725 (2005).</li><li>Ernst, O., Vayttaden, S. J., Fraser, I. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+NF-kappaB+Activation+in+TLR-Activated+Macrophages.">Measurement of NF-kappaB Activation in TLR-Activated Macrophages.</a> Methods in Molecular Biology. 1714, 67-78 (2018).</li><li>Rodriguez, M. S., Thompson, J., Hay, R. T., Dargemont, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+retention+of+IkappaBalpha+protects+it+from+signal-induced+degradation+and+inhibits+nuclear+factor+kappaB+transcriptional+activation.">Nuclear retention of IkappaBalpha protects it from signal-induced degradation and inhibits nuclear factor kappaB transcriptional activation.</a> Journal of Biological Chemistry. 274 (13), 9108-9115 (1999).</li><li>Mendez, J. M., Kolora, L. D., Lemon, J. S., Dupree, S. L., Keestra-Gounder, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+endoplasmic+reticulum+stress+response+impacts+the+NOD1+signaling+pathway.">Activation of the endoplasmic reticulum stress response impacts the NOD1 signaling pathway.</a> Infection and Immunity. , (2019).</li><li>Hoiseth, S. K., Stocker, B. A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aromatic-Dependent+Salmonella-Typhimurium+Are+Non-Virulent+and+Effective+as+Live+Vaccines.">Aromatic-Dependent Salmonella-Typhimurium Are Non-Virulent and Effective as Live Vaccines.</a> Nature. 291 (5812), 238-239 (1981).</li><li>Keestra, 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=Manipulation+of+small+Rho+GTPases+is+a+pathogen-induced+process+detected+by+NOD1.">Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1.</a> Nature. 496 (7444), 233-237 (2013).</li><li>Wong, D., 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=Extensive+characterization+of+NF-kappaB+binding+uncovers+non-canonical+motifs+and+advances+the+interpretation+of+genetic+functional+traits.">Extensive characterization of NF-kappaB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.</a> Genome Biology. 12 (7), R70 (2011).</li><li>Gupta, R., 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=Firefly+luciferase+mutants+as+sensors+of+proteome+stress.">Firefly luciferase mutants as sensors of proteome stress.</a> Nature Methods. 8 (10), 879-884 (2011).</li><li>Cogswell, J. 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The major contribution of the protocol described is that it provides a fast and easy method to detect NF-&#954;B activation in cells, which allows for high throughput analysis of multiple stimulatory conditions or drugs affecting NF-&#954;B activation. Here, we describe a protocol for NF-&#954;B activation in Salmonella-infected HeLa cells. These cells can be used for infection with other pathogens as well to study the impact of bacterial infection on NF-&#954;B activation. In addition, NF-&#954;B-dependent luci...

The nuclear factor-&#954;B (NF-&#954;B) family of proteins are important transcription activators that regulate gene expression in various biological pathways. Activation of NF-&#954;B induces transcription of target genes, many of which are important for immune and inflammatory responses, cell proliferation, stress responses and cancer progression1,2. NF-&#954;B plays an integral role in mediating early inflammatory outcomes for pathogen clearance. Given the many biological processes mediated by NF-&#954;B activation, disruptions in its signaling can have serious consequences for health and disease. Loss of function mutations in NF-&#954;B signaling are associated in several immune deficiency phenotypes, while gain of function mutations are associated with several types of cancers, including B-cell lymphomas and breast cancers3. Additionally, many pathogens have been shown to directly modulate the activation state of NF-&#954;B through expression of virulence factors4,5,6,7.
Activation of NF-&#954;B is known to be a consequence of many variable stimuli including bacterial products such as lipopolysaccharides (LPS), flagellin and peptidoglycans known as pathogen-associated molecular patterns (PAMPs). These PAMPs are detected by pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) and Nod-like receptors (NLRs) leading to the activation of NF-&#954;B and the subsequent expression of an array of NF-&#954;B-dependent inflammatory genes8. In addition to PRR activation by PAMPs, other bacterial products, such as bacterial effector proteins, can induce the activation of NF-&#954;B. Interestingly, bacteria also express effector proteins that actively attenuate the NF-&#954;B pathway and enhance their pathogenicity, underscoring the importance of NF-&#954;B as an essential mediator of immunity9.
There are five different subunits that form the NF-&#954;B dimers; p50, p52, RelA (p65), RelB and cRel. The two main NF-&#954;B heterodimers are the p50:RelA and the p52:RelB dimers. The activated NF-&#954;B dimers bind to DNA sites, known as &#954;B sites, in the promoter and enhancer regions of various target genes. Under normal homeostatic conditions, NF-&#954;B interacts with a family of inhibitor proteins known as I&#954;B proteins to remain inactive. Upon stimulation, I&#954;B is phosphorylated by I&#954;B Kinase (IKK), which allows it to be targeted for ubiquitination, and subsequently degradation. Degradation of I&#954;B activates NF-&#954;B by revealing a nuclear localization signal. NF-&#954;B then translocates to the nucleus, where it binds &#954;B sites in the promoter region of target genes and promote transcription10. Thus, activation of NF-&#954;B upregulates mRNA expression of NF-&#954;B target genes, and this change can be measured through RNA quantification assays such as RT-qPCR11.
Several methods exist and are commonly used for the measurement of NF-&#954;B activation, including electrophoretic mobility shift assays (EMSA), nuclear translocation, and gene reporter assays. EMSA is used to detect protein complexes with nucleic acids. Stimulated cells are fractionated to isolate nuclear proteins, including the translocated NF-&#954;B, which is then incubated with radiolabeled nucleotides containing the NF-&#954;B binding domain. The samples are run on a gel and imaged by autoradiography of 32P-labeled nucleic acid. If NF-&#954;B is present in the protein fraction, it will bind the nucleotides, which will migrate slower through the gel and present as discrete bands. Nuclear fractions of cells lacking activated NF-&#954;B (e.g., unstimulated control cells) will produce no bands as the nucleotides migrate faster to the end of the gel. A major drawback of this method is that it is largely quantitative in the binary sense (i.e., on or off) and does not adequately capture meaningful differences in NF-&#954;B binding capacity. Additionally, this method does not consider chromatin structures that are functionally important for NF-&#954;B target genes12,13.
Similar to the previous method, there is a &#34;non-shift&#34; assay in which multi-well plates are coated with nucleotides containing the NF-&#954;B binding sequence. Following treatment of cells with nuclear fractions of protein, NF-&#954;B will bind to the nucleotides bound to the well. Anti-NF-&#954;B antibodies are then added, which will interact with the bound NF-&#954;B and produce a colorimetric signal proportional to the amount of NF-&#954;B, indicating the degree of NF-&#954;B activation. This method is advantageous over the EMSA in that it does not require radiolabeled nucleic acids and is quantitative, in comparison. However, a caveat of this method is that it again does not differentiate between chromatin states of NF-&#954;B target genes14.
Another method by which NF-&#954;B activation may be detected is by chromatin immunoprecipitation (ChIP), whereby DNA and interacting proteins are cross-linked with formaldehyde and immunoprecipitated with specific anti-NF-&#954;B antibodies. The specific nucleotide fragments are then purified and identified through PCR amplification or direct high throughput sequencing. Results generated from this method provide semi-quantitative results of NF-&#954;B binding activity with target genes. However, the results are highly dependent on the fixation conditions and purification processes at each step15.
In nuclear translocation assays, cells are stimulated to induce NF-&#954;B activation and then fixed. Anti-p65 antibodies are added to fixed cells. Alternatively, the p65 subunit itself can be tagged with a fluorescent peptide such as green fluorescent green (GFP). In either case, immunofluorescence will allow imaging of localization of p65 to determine cellular distribution. By measuring the proportion of cytosolic and nuclear localized protein, investigators can determine the relative activation state of NF-&#954;B. A drawback of this method is that the immunofluorescence is comparatively time consuming, requires expensive antibodies, and needs relatively greater technical expertise16.
Reporter genes are commonly used tools to study the regulatory and expression patterns of a gene of interest. Typically, reporter genes are constructed from the promoter sequence of a gene of interest fused to a gene coding for an easily detectable protein. Proteins with enzymatic activities, fluorescence, or luminescence properties are commonly chosen for their ability to be assayed and quantified. Thus, the read-out (e.g., luminescence, fluorescence) serves as a signal for detection of the gene expression. These reporter constructs can then be introduced into different cell types, such as epithelial cells or macrophages.
Described in the protocol is the use of a cloned HeLa cell line (HeLa 57A) that is stably transfected with a luciferase reporter containing three copies of the &#954;B consensus of the immunoglobulin &#954;-chain promoter region17. Expression of luciferase is dependent on the activation of NF-&#954;B, which occurs following cell stimulation. Stimulated cells are easily lysed using cell lysis buffer provided in the luciferase assay kit. A portion of the cell lysate is then mixed with luciferase assay buffer that contains luciferin. Luciferin is the substrate of luciferase and is required for the generation of light in the presence of luciferase. After combining the assay buffer with the lysate, the solution will emit light in a process known as luminescence. The amount of light produced, given in lumens, is proportional to the amount of luciferase present in the lysate and serves as a measure of NF-&#954;B activation. The lumen readings are interpreted in comparison to an unstimulated standard to account for baseline NF-&#954;B activity and the signal itself is stable for several minutes to allow for reliable measurement. In addition, the HeLa 57A cell line is stably transfected with a NF-&#954;B-independent &#946;-galactosidase reporter. The &#946;-galactosidase reporter is constitutively expressed, and &#946;-galactosidase activity can be measured to control for cell viability or variation in cell numbers17. The luciferase values can then be adjusted to the &#946;-galactosidase values and reported as fold increase over the unstimulated control cells.
Since NF-&#954;B is a transcription factor responsible for the increased expression of NF-&#954;B-dependent target genes, a follow up experiment to control for NF-&#954;B-dependent increased gene expression is quantitative reverse transcription polymerase chain reaction (RT-qPCR). RT-qPCR is a highly sensitive method by which changes in the gene expression can be quantified over several orders of magnitude. Stimulated and control cells are harvested for RNA via phenol-chloroform extraction. Following phase separation, RNA is extracted as the major component of the aqueous layer. RNA is then precipitated and washed to produce a pure pellet. This pellet is then reconstituted and further cleaned of contaminant DNA via DNase treatment. The pure RNA is then reverse transcribed to create complementary DNA (cDNA). This cDNA can then be analyzed through quantitative PCR techniques, where the abundance of a specific mRNA sequence is quantified to determine gene expression. This technique does not elucidate translational control, post translational modification, protein abundance, or protein activity. However, many genes, particularly those involved in pro-inflammatory processes, are regulated via NF-&#954;B and their mRNA abundance is indicative of their expression.
The method proposed here utilizes a fast and simple way by which NF-&#954;B activation can be detected via luminescence assays of cellular lysate. RT-qPCR of NF-&#954;B target gene expression can be used to quantify expression of particular genes, as well as validate functional activity of NF-&#954;B activation. The major advantages of such a system are its simplicity and speed, which allows for high throughput screening of a range of conditions that modulate NF-&#954;B activation. This protocol is suitable for other cell lines expressing an NF-&#954;B::luciferase reporter, and has been demonstrated in stably transfected RAW264.7 cells18. The amount of time required to handle samples, starting from cell lysis to generating a luminescence signal, is minimal and takes the span of about an hour. Measurement of NF-&#954;B requires only basic laboratory equipment such as opaque plates, a plate reader capable of measuring luminescence, and simple data analysis software such as a spreadsheet program.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesive film</td>
			<td>VWR International</td>
			<td>60941-070</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Fisher Bioreagents</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>11665092</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse treatment kit</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Promega</td>
			<td>U1511</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Fisher Bioreagents</td>
			<td>BP2818100</td>
			<td>molecular grade</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa 57A cells</td>
			<td></td>
			<td></td>
			<td>Ref # 15</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropanol</td>
			<td>Fisher Bioreagents</td>
			<td>BP26181</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Bioreagents</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Fisher Bioreagents</td>
			<td>BP1425-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny broth</td>
			<td>Fisher Bioreagents</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Chemical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>spectrophotometer</td>
		</tr>
		<tr>
			<td>promega luciferase assay system</td>
			<td>Promega</td>
			<td>E1501</td>
			<td>Cell lysis buffer &#38; luciferin substrate</td>
		</tr>
		<tr>
			<td>Random Hexamers</td>
			<td>Thermo Scientific</td>
			<td>SO142</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time GAPDH forward primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-CCAGGAAATGAGCTTGAC 
			AAAGT-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time GAPDH reverse primer</td>
			<td></td>
			<td></td>
			<td>5-&#39;CCCACTCCTCCACCT 
			TTGAC-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-23 forward primer</td>
			<td></td>
			<td></td>
			<td>5-&#39;GAGCCTTCTCTGCTCCC 
			TGAT-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-23 reverse primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-AGTTGGCTGAGGCCCAGTAG-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-6 forward primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-GTAGCCGCCCCACACAGA-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-6 reverse primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-CATGTCTCCTTTCTCAGG 
			GCTG-3&#39;</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>Applied Biosystems</td>
			<td>4308228</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td></td>
		</tr>
		<tr>
			<td>RT buffer</td>
			<td>Promega</td>
			<td>A3561</td>
			<td></td>
		</tr>
		<tr>
			<td>SL1344</td>
			<td></td>
			<td></td>
			<td>Ref # 17</td>
		</tr>
		<tr>
			<td>SL1344 &#916;sipA sopB::MudJ sopE2::pSB1039</td>
			<td></td>
			<td></td>
			<td>Ref # 18</td>
		</tr>
		<tr>
			<td>SL1344 &#916;sopE &#916;sipA sopB::MudJ sopE2::pSB1039</td>
			<td></td>
			<td></td>
			<td>Ref # 18</td>
		</tr>
		<tr>
			<td>SYBR green</td>
			<td>Applied Biosystems</td>
			<td>4309155</td>
			<td>2x mastermix</td>
		</tr>
		<tr>
			<td>Tri-reagent</td>
			<td>Molecular Research Center</td>
			<td>TR 118</td>
			<td>guanidine thiocyanate</td>
		</tr>
		<tr>
			<td>Trypsin -EDTA</td>
			<td>Thermo Fisher</td>
			<td>25300054</td>
			<td>0.05% Trypsin-EDTA</td>
		</tr>
		<tr>
			<td>ultrapure water</td>
			<td>Fisher Bioreagents</td>
			<td>BP248450</td>
			<td></td>
		</tr>
		<tr>
			<td>Well plate for PCR</td>
			<td>VWR International</td>
			<td>89218-294</td>
			<td>384-well plate</td>
		</tr>
	</tbody>
</table>

nf-&#954;b-dependent luciferase activation and quantification of gene expression in salmonella infected tissue culture cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

The assay described here focuses on activation of the transcription factor NF-&#954;B using a NF-&#954;B-dependent luciferase reporter that is stably transfected into a line of HeLa cells. Activated NF-&#954;B translocates to the nucleus where it binds &#954;B binding sites of target genes, including the pro-inflammatory cytokines IL6 and IL23. A general overview of NF-&#954;B activation is depicted in Figure 1. The gram-negative bacterium <...

Watch this Scientific Journal Video about NF-ÎºB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells at JoVE.com

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

This protocol is useful for determining changes in NF-kappa-B activation in cells expressing an NF-kappa-B luciferase reporter. The main advantage of this technique is it allows for a high-throughput screen of factors or conditions that lead to changes in NF-kappa-B activation. NF-kappa-B is a transcription factor for a wide variety of cellular processes.A well known function of NF-kappa-B is in the induction of inflammatory responses by inducing the expression of various cytokines and chemokines. Our lab is particularly interested in the induction of NF-kappa-B induced by the pathogen Salmonella typhimurium. Here, we use the HeLa cell line that is stably transfected with an NF-kappa-B luciferase reporter plasmid.Other bacteria, or even viruses or chemical compounds, can be used in this cell line to study the activation of NF-kappa-B. Other cell lines can also be used if you can introduce such a NF-kappa-B luciferase reporter plasmid in that cell line. Somebody doing this for the first time may have issues properly utilizing sterile technique that is required for cell culture and bacterial work.One day before cell stimulation, remove the growth media of HeLa 57A cells that have been grown to about 75%confluency. Wash cells with one milliliter of 0.05%trypsin EDTA. Replace with another milliliter of trypsin EDTA and transfer the flask to a 37 degrees Celsius incubator for five minutes.After the cells have been detached from the flask, suspend them in 10 milliliters of growth media. Combine 10 microliters of cell suspension with 10 microliters of Trypan blue, pipette up and down to mix, and transfer 10 microliters to a cell counter slide. Count cells in suspension using a cell counter, and use growth media to dilute the cells to a final concentration of 2.5 times 10 to the five cells per milliliter in a 50 milliliter conical tube.Transfer 250 microliters of the cell suspension to each well of a 48-well plate. Periodically cap and turn over the conical tube to ensure a homogenous cell suspension. Tap the plate gently on the side to ensure that the cells distribute uniformly in the wells.Transfer the plate to 37 degrees Celsius in a 5%carbon dioxide incubator and allow the cells to attach and grow overnight. Streak frozen stocks of Salmonella onto LB agar plates to produce single colonies. Transfer the plates to an incubator set to 37 degrees Celsius and allow overnight growth.The next day, add three milliliters of LB to sterile bacterial culture tubes, and add appropriate antibiotics to the media. Using a sterile inoculation loop, pick a single colony from the streaked bacterial cultures and touch the loop to the LB media. Cap the tubes after inoculating and discard the loop.Place the tubes in a shaking incubator set at 37 degrees Celsius and 180 RPM, then allow the bacteria to grow overnight. In the morning, retrieve overnight bacterial cultures from the incubator. Prepare tubes for subculture by adding three milliliters of fresh LB and antibiotics.Transfer 30 microliters of the overnight bacterial culture to the freshly prepared media. Place the tubes in a shaking incubator set at 37 degrees Celsius for three hours. After the three hour incubation, transfer one milliliter of sterile LB broth into a plastic cuvette to serve as the blank.Transfer 900 microliters of LB into the other cuvettes to be used for the sample analysis. Transfer 100 microliters of bacterial subculture into a cuvette containing 900 microliters of LB, and pipette up and down several times to mix. Repeat this for each bacterial suspension.Turn on the spectrophotometer to measure the optical density of the bacterial cultures at a wavelength of 600 nanometers. Place the blank in the spectrophotometer. Take note of the orientation.Close the lid and press the blank button on the spectrophotometer to get the background absorbance. Replace the blank cuvette with a sample cuvette in the same orientation and press read. Record the OD600 values of these samples.Multiply the value by 10 to account for the dilution factor. Dilute the suspension one to 10 in a cuvette and measure absorbance, which should give a value of approximately 0.1 which roughly corresponds to one times 10 to the nine CFU per milliliter. In a new tube, add an appropriate volume of the diluted subculture to fresh LB to achieve a suspension of one times 10 to the eight CFU per milliliter to be used as an inoculum.Prepare serial dilutions of the inoculum by transferring 50 microliters of bacterial suspension to a tube containing 450 microliters of sterile PBS until a final dilution of approximately 100 CFU per milliliter is made. Transfer 100 microliters of the two lowest dilutions, 100 and 1000 CFU per milliliter, to two LB agar plates, and spread the suspension with a cell spreader to obtain single colonies. Transfer these plates to a 37 degree Celsius incubator and incubate overnight.The following day, count the colonies and calculate the bacterial concentration of the initial inoculum to determine the actual inoculum concentration. On the same day, label the lid of the plate according to the infection conditions that will be used for each well, with each condition being done in triplicate. Add 10 microliters of the inoculum to appropriate wells, and add 10 microliters of sterile LB to uninfected control wells.To synchronize the time of infection, place the plate in a tabletop centrifuge and spin at 500 times G for five minutes, ensuring that the plate is counterbalanced. Then, transfer the infected cells to a 5%carbon dioxide incubator at 37 degrees Celsius for one hour. Place a 15 milliliter conical tube containing an aliquot of cell culture media in a 37 degrees Celsius water bath for use in the next step.One hour after the time of infection, remove the 15 milliliter conical tube containing cell culture media from the water bath and wipe the exterior with 70%ethanol. Transfer the tissue culture plates from the incubator to a biosafety cabinet. Use sterile tips to aspirate media from the wells and replace with 250 microliters of fresh, warm cell culture media.Return the plates to the 5%carbon dioxide incubator at 37 degrees Celsius for an additional four hours. After that, remove the plates from the carbon dioxide incubator and aspirate the media from the wells. For luciferase analysis, add 100 microliters of 1X cell lysis buffer to the wells.Transfer the plate to a negative 80 degrees Celsius freezer and incubate for at least 30 minutes to ensure efficient cell lysis. Then, place the plate containing frozen cell lysate on a bench to thaw, and prepare luciferase substrate reagents according to manufacturer recommendations. Allow luciferase substrate reagents to equilibrate to room temperature.Next, turn on the plate reader and open the corresponding reader program. Set the machine to measure luminescence. Transfer 10 microliters of each cell lysate to a well of an opaque 96-well plate.Using a multichannel pipette, add 50 microliters of the luciferase assay reagent to each well of the opaque plate. Gently tap the plate on the side to mix wells, and ensure that the bottom surface is covered with liquid. Place the plate in a plate reader and initiate reading.Copy the luminescence values into a spreadsheet program and plot the results. This protocol focuses on the activation of the transcription factor NF-kappa-B using an NF-kappa-B dependent luciferase reporter that is stably transfected into a line of HeLa cells. This figure shows a representative experiment of NF-kappa-B dependent luciferase activation and IL6 gene expression in HeLA 57A cells infected with the Salmonella typhimurium strains.Infection with the wild type SL1344 strain induced a strong luciferase signal in both the RLU and IL6 expression, which was decreased in cells infected with the sipA sopB sopE2 triple mutant and reduced to control levels with the sipA sopB sopE2 sopE quadruple mutant. Pay close attention when doing your serial dilutions and plating. This will ensure that you have a consistent inoculum across your strains.After identifying factors that contribute to NF-kappa-B activation and downstream changes in mRNA expression, western blot technique can be used to identify changes in protein expression. This protocol has allowed our lab not only to evaluate NF-kappa-B activation induced by Salmonella but other compounds and proteins implicated in NF-kappa-B activation. Salmonella typhimurium is a human pathogen.Employ proper PPE when working with these bacteria.

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