This work was supported by the Bill and Melinda Gates Foundation (Grant 53085), the JR Oishei Foundation, and The American Lung Association (Grant RG-123721-N) to APH, and NIH (NIDCD) fellowship F31DC011218 to EAC.

1. Preparing Bacterial&#160;Cultures
<ol>
	<li>Growing culture for use in experiments
	<ol>
		<li>In a 37 &#176;C heating block, thaw a frozen stock-vial of S. pneumoniae and add its content to 9 ml of fresh, prewarmed Todd-Hewitt broth with 0.5% yeast extract (THY) for a total volume of 10 ml in a glass culture tube.</li>
		<li>Incubate statically at 37 &#176;C until the culture reaches mid-log phase (Abs600nm &#8776;0.5-0.6).</li>
	</ol>
	</li>
</ol>
2. Detecting Membrane Depolarization and Rupture
<ol>
	<li>Preparing reagents
	<ol>
		<li>Prepare a 50 &#181;M stock of DiBAC4(3) in 100% DMSO, and a 2 mg/ml stock of PI in ddH2O. The DiBAC4(3) stock is stable at -20 &#176;C for at least 6 months. The PI stock is stable at 4 &#176;C for at least 6 months.</li>
		<li>Wrap stocks in foil to protect from the light.</li>
	</ol>
	</li>
	<li>Loading bacteria
	<ol>
		<li>Pellet the bacterial cells from step 1.2.2 by centrifugation at 2,400 x g for 10 min at room temperature and wash twice by removing the supernatant, resuspending the pellet in 10 ml of 1x Phosphate-Buffered Saline (PBS), and centrifuge again at 2,400 x g for 10 min.</li>
		<li>Remove supernatant and resuspend pellet in PBS to the original volume. This will provide for approximately 108 colony-forming units of bacteria per ml. Remove 1 ml (enough for 5 samples) of the washed cells and place into a microcentrifuge tube.</li>
		<li>To these cells, add 25 &#181;l of a 1 M filter-sterilized stock solution of glucose (prepared in ddH2O and filtered through a 0.45 &#181;m filter) for a final concentration of 25 mM glucose, and incubate in a 37 &#176;C heating block for fifteen min. NOTE: This provides energy for all ATP-dependent membrane channels and cellular components. The need for this step may vary, depending on the experiment.</li>
		<li>Add 5 &#181;l of the 50 &#181;M DiBAC4(3) stock and 10 &#181;l of the 2 mg/ml PI stock. This provides for final concentrations of 250 nM and 20 &#181;g/ml, respectively. Mix thoroughly by pipetting up and down several times. Add 200 &#181;l&#160;of this cell suspension to each sample well of a clear 96-well plate.</li>
	</ol>
	</li>
	<li>Detecting fluorescence
	<ol>
		<li>Place plate in a prewarmed (37 &#176;C) fluorescence detection plate reader.</li>
		<li>Ensure that appropriate filters are in place for DiBAC4(3) (490 nm excitation, 516 nm emission) and PI (535 nm excitation, 617 nm emission) detection.</li>
		<li>To allow for (and concurrently monitor) equilibration of DiBAC4(3) over the membrane, set the reader to take fluorescence measurements every min for about 30-40 min, or until reading stabilizes; this serves as a &#8220;pretreatment&#8221; reading.</li>
		<li>Eject plate and add the experimental agent of choice and immediately place the plate back in the reader to continue monitoring DiBAC4(3) and PI fluorescence for the desired length of time. If treating several samples at once, using a multichannel pipette can be helpful to add the agent simultaneously to all wells.</li>
		<li>Export the readings to a data analysis program such as Microsoft Excel. Plot fluorescence over time to evaluate membrane depolarization and rupture. Increasing fluorescence of DiBAC and PI indicates that depolarization and rupture are occurring.</li>
	</ol>
	</li>
</ol>
3. Detecting Changes in Intracellular Ca2+ or K+ Concentrations
<ol>
	<li>Preparing reagents and loading buffer
	<ol>
		<li>Prepare the following reagents: 5 mM stock of the ion-sensitive fluorescent dye (Fura-2/AM or PBFI/AM; add the appropriate amount of DMSO to a tube containing 50 &#181;g of the dye), &#8220;100x&#8221; probenecid (add 1 ml of PBS to a 77 mg vial). The solubilized dyes can be frozen at -20 &#176;C once and are stable in solution for 3 months, whereas the probenecid is stable in solution at -20 &#176;C for 6 months.</li>
		<li>Prepare 1 ml of 2x Loading Buffer as follows: In the bottom of a 15 ml plastic conical tube, dispense 20 &#181;l&#160;of 100x PowerLoad concentrate (stable at 2-8 &#176;C for 6 months), followed by 2 &#181;l&#160;of the 5 mM ion-sensitive dye directly into the PowerLoad. Vortex briefly to mix. Add 960 &#181;l&#160;of PBS followed by 20 &#181;l&#160;of 100x probenecid. Vortex the mixture one final time. Wrap in foil to protect from light.</li>
	</ol>
	</li>
	<li>Loading the bacteria with dye
	<ol>
		<li>Pellet and wash the mid-log phase bacterial culture as described in 2.2.1. Remove supernatant and resuspend pellet in 5 ml of PBS to make a &#8220;2x&#8221; cell suspension.</li>
		<li>Add 1 ml of the 2x cell suspension to the 2x Loading Buffer. This will provide for a final volume of 2 ml, and final concentrations of 1x PowerLoad, 5 &#181;M fluorescent dye, and 1x probenecid. NOTE: This suspension provides enough volume for 10 samples since each sample well for measurement will contain 200 &#181;l.</li>
		<li>Incubate in a 37&#160;&#176;C water bath for 75 min, protected from light. NOTE: The dye is loaded into the bacterial cells in the absence of any glycolytic substrates and in the presence of probenecid to minimize efflux of the dye.</li>
		<li>WASH 1:&#160; Pellet the cells by centrifugation at 2,400 x g for 10 min, remove supernatant, and resuspend in 2 ml PBS containing 1x probenecid. WASH 2, 3:&#160; Repeat step 3.2.4&#160;2x. Incubate at 37&#160;&#176;C for another 30 min. This will&#160;allow time for bacterial esterases to hydrolyze the AM ester group away from the internalized fluorescent dyes.</li>
		<li>WASH 4: Pellet the cells by centrifugation at 2,400 x g for 10 min, remove supernatant, and resuspend in 2 ml of PBS containing 1x probenecid. NOTE: Depending on the experiment, glucose can be added back here to re-energize the bacteria.</li>
	</ol>
	</li>
	<li>Detecting fluorescence
	<ol>
		<li>Prewarm the fluorescence detection plate-reader plate reader to 37 &#176;C.</li>
		<li>For each desired sample, add 200 &#181;l&#160;of the loaded cells (from step 3.2.8) to the wells of a 96-well plate. (Perform the following steps to completion for one well/sample at a time).</li>
		<li>To establish a baseline reading for a well, place plate in the fluorescence detection plate-reader to measure the fluorescence (340 nm excitation / 510 nm emission for &#8220;Value 1&#8221; and 380 nm excitation / 510 nm emission for &#8220;Value 2&#8221;) at every sec for one min.</li>
		<li>Eject plate, add the treatment of choice to that well (in a small volume of approximately 5 &#181;l), quickly pipette up and down a few times to mix, and immediately place back in the fluorescence detection plate-reader to read every sec for desired length of time. (If the injectors associated with the plate reader are available, these could also be used to seamlessly add the desired treatment, eliminating the need to stop the read and eject the plate).</li>
		<li>Export the readings to a data analysis program such as Microsoft Excel. Calculate the ratio of the fluorescence values for each time-point by dividing Value 1 by Value 2, and plot the ratio values on a graph.</li>
	</ol>
	</li>
</ol>
4. Detecting Changes in Intracellular pH
<ol>
	<li>Preparing reagents and calibration buffers
	<ol>
		<li>Prepare the following reagents: 5 mM stock of BCECF/AM (add the appropriate amount of DMSO to a tube containing 50 &#181;g of the dye. This stock is stable at -20 &#176;C for 3 months); 100x probenecid (add 1 ml of PBS to a 77 mg vial); 1 mM stock preparation of nigericin in ethanol (required to equilibrate the intracellular pH (pH&#173;i) of the cells &#173;to the pH of the surrounding buffer. This stock is stable at -20 &#176;C for 6 months); 0.5 mM stock of Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in DMSO (protonophore to uncouple the proton motive force. This stock is stable at -20 &#176;C for 6 months)</li>
		<li>Prepare 10 ml of the following high potassium buffers: 135 mM KH2PO4 / 20 mM NaOH (monobasic) and 110 mM K2HPO4 / 20 mM NaOH (dibasic). Filter sterilize using a 0.45 &#181;m filter. (These buffers can be stored at 4 &#176;C until use).</li>
		<li>Mix the high potassium buffers at the necessary proportions to create high potassium buffers with at least 5-6 pH values ranging from 6.5 to 8.0.</li>
		<li>Prepare 1 ml of 2x Loading Buffer as follows: In the bottom of a 15 ml plastic conical tube, dispense 40 &#181;l&#160;of 100x&#160;PowerLoad, followed by 20 &#181;l&#160;of the 5 mM ion-sensitive dye directly into the PowerLoad. Vortex briefly to mix. Add 900 &#181;l&#160;of PBS followed by 40 &#181;l&#160;of 100x probenecid. Vortex the mixture one final time. Wrap in foil to protect from light.</li>
	</ol>
	</li>
	<li>Loading the bacteria with dye
	<ol>
		<li>Pellet and wash the 8 ml of the mid-log phase bacterial culture as described in 2.2.1. Remove supernatant, resuspend pellet in 2 ml of PBS to make a &#8220;4x&#8221; cell suspension.</li>
		<li>Add 1 ml of the 4x&#160;cell suspension to the 2x&#160;Loading Buffer. This will provide for a final volume of 2 ml, and final concentrations of 2x&#160;cells, 2x&#160;PowerLoad, 50 &#181;M fluorescent dye, and 2x probenecid.</li>
		<li>Incubate in a 30 &#176;C water bath for 40 min, protected from light. NOTE: The dye is loaded into the bacterial cells at the lower temperature of 30 &#176;C and in the absence of any glycolytic substrates to minimize efflux of the dye.</li>
		<li>WASH 1:&#160; Pellet the cells by centrifugation at 2,400 x g for 10 min, remove supernatant to get rid of excess dye, and resuspend in 4 ml PBS containing 2x probenecid and 1 mM glucose (to reenergize the bacteria). WASH 2, 3:&#160; Repeat step 4.2.7 twice, with final resuspension in 4 ml&#160;PBS (for a final concentration of 1x&#160;cells) containing 2x probenecid and 10 mM glucose.</li>
		<li>Incubate at 37 &#176;C for 5 min. This will allow the cells to fully energize and allow time for bacterial esterases to cleave the AM ester group away from the internalized BCECF.</li>
	</ol>
	</li>
	<li>Establishing background fluorescence and in vivo calibration curve
	<ol>
		<li>Prewarm the fluorescence detection plate-reader plate reader to 37 &#176;C.</li>
		<li>To determine background fluorescence, filter-sterilize about 500 &#181;l&#160;of the loaded and energized bacterial suspension (from step 4.2.7) to remove the bacteria, and add 200 &#181;l&#160;of filtrate to the well of the 96-well plate.</li>
		<li>Place plate in the fluorescence detection plate-reader to measure the fluorescence (490 nm excitation / 530 nm emission for &#8220;Value 1&#8221; and 440 nm excitation / 530 nm emission for &#8220;Value 2&#8221;) for 1 min. This background fluorescence should be subtracted before calculation of the ratios for both the calibration curve and the experimental samples.</li>
		<li>To obtain an in vivo calibration curve, wash 500 &#181;l&#160;aliquots of the loaded and energized cells from step 4.2.7 once and resuspend in high potassium buffers at different pH values (ranging from 6.5-8.0).</li>
		<li>Add 20 &#181;M nigericin to the samples to equilibrate the intracellular pH of the cells to the pH of the surrounding buffer and incubate at 37 &#176;C for 5 min. Add 200 &#181;l&#160;of bacteria/pH buffer combinations to the wells of a 96-well plate.</li>
		<li>Place plate in the fluorescence detection plate-reader to measure the fluorescence for a few min to establish a steady reading for each well. Export the readings to a data analysis program such as Microsoft Excel.</li>
		<li>After subtracting the background fluorescence values, calculate the ratio of the fluorescence values by dividing Value 1 by Value 2. Plot the ratio values for each pH buffer to create the calibration curve. (A calibration curve should be generated for every new batch of loaded cells, as the amount of fluorescent dye that is loaded into the bacteria can vary each time).</li>
	</ol>
	</li>
	<li>Measuring changes in intracellular pH
	<ol>
		<li>For each desired sample, add 200 &#181;l&#160;of the loaded and energized cells (from step 4.2.7) to the wells of a 96-well plate.</li>
		<li>Place plate in the fluorescence detection plate-reader and measure the fluorescence of the samples every 5 sec for 5 min.</li>
		<li>Eject the plate and add 10 &#181;M CCCP to one well (serves as the positive control for decreasing intracellular pH) and the experimental agent(s) of interest to the other wells. To accurately compare their effects over time, add the CCCP and agents of choice at the same time using a multichannel pipette.</li>
		<li>Immediately return the plate to the plate-reader and continue measuring fluorescence every 5 sec for 10 min. As an additional control, eject the plate and add 20 &#181;M Nigericin to all samples to equilibrate the pH&#173;i of the bacteria &#173;to the pH of the surrounding buffer, and read for 5 min.</li>
		<li>After subtracting the background fluorescence values, calculate the ratios of fluorescence for each sample. Interpolate the pHi for each reading from the calibration curve.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hakansson, A., Zhivotovsky, B., Orrenius, S., Sabharwal, H., Svanborg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+induced+by+a+human+milk+protein.">Apoptosis induced by a human milk protein.</a> Proc. Natl. Acad. Sci. U.S.A. 92 (17), 8064-8068 (1995).</li><li>Hakansson, A., 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=A+folding+variant+of+alpha-lactalbumin+with+bactericidal+activity+against+Streptococcus+pneumoniae.">A folding variant of alpha-lactalbumin with bactericidal activity against Streptococcus pneumoniae.</a> Mol. Microbiol. 35 (3), 589-600 (2000).</li><li>Hakansson, A. P., Roche-Hakansson, H., Mossberg, A. K., Svanborg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-Like+Death+in+Bacteria+Induced+by+HAMLET,+a+Human+Milk+Lipid-Protein+Complex.">Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex.</a> PLoS One. 6 (3), (2011).</li><li>Kohler, C., Gogvadze, V., Hakansson, A., Svanborg, C., Orrenius, S., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+folding+variant+of+human+alpha-lactalbumin+induces+mitochondrial+permeability+transition+in+isolated+mitochondria.">A folding variant of human alpha-lactalbumin induces mitochondrial permeability transition in isolated mitochondria.</a> Eur. J. Biochem. 268 (1), 186-191 (2001).</li><li>Ly, J. D., Grubb, D. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+membrane+potential+(&#916;&#968;+m)+in+apoptosis;+an+update.">The mitochondrial membrane potential (&#916;&#968; m) in apoptosis; an update.</a> Apoptosis. 8 (2), 115-128 (2003).</li><li>Dominguez, 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=Calcium+signalling+in+bacteria.">Calcium signalling in bacteria.</a> Mol. Microbiol. 54 (2), 291-297 (2004).</li><li>Corratge-Faillie, C., Jabnoune, M., Zimmermann, S., Very, A. A., Fizames, C., Sentenac, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+and+sodium+transport+in+non-animal+cells:+the+Trk/Ktr/HKT+transporter+family.">Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family.</a> Cell Mol. Life Sci. 67 (15), 2511-2532 (2010).</li><li>Szabo, I., Petronilli, V., Zoratti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+patch-clamp+study+of+Bacillus+subtilis.">A patch-clamp study of Bacillus subtilis.</a> Biochim. Biophys. Acta. 1112 (1), 29-38 (1992).</li><li>Zoratti, M., Petronilli, V., Szabo, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-activated+composite+ion+channels+in+Bacillus+subtilis.">Stretch-activated composite ion channels in Bacillus subtilis.</a> Biochem. Biophys. Res. Commun. 168 (2), 443-450 (1990).</li><li>Novo, D., Perlmutter, N. G., Hunt, R. H., Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+flow+cytometric+membrane+potential+measurement+in+bacteria+using+diethyloxacarbocyanine+and+a+ratiometric+technique.">Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.</a> Cytometry. 35 (1), 55-63 (1999).</li><li>Novo, D. J., Perlmutter, N. G., Hunt, R. H., Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+flow+cytometric+analysis+of+antibiotic+effects+on+membrane+potential,+membrane+permeability,+and+bacterial+counts+of+Staphylococcus+aureus+and+Micrococcus+luteus.">Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus.</a> Antimicrob. Agents Chemother. 44 (4), 827-834 (2000).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+potential+estimation+by+flow+cytometry.">Membrane potential estimation by flow cytometry.</a> Methods. 21 (3), 271-279 (2000).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+analysis+at+the+single-cell+level:+tasks+and+techniques.">Microbial analysis at the single-cell level: tasks and techniques.</a> J. Microbiol. Methods. 42 (1), 3-16 (2000).</li><li>Bashford, C. L., Chance, B., Smith, J. C., Yoshida, 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+behavior+of+oxonol+dyes+in+phospholipid+dispersions.">The behavior of oxonol dyes in phospholipid dispersions.</a> Biophys. J. 25 (1), 63-85 (1979).</li><li>Suzuki, H., Wang, Z. -. Y., Yamakoshi, M., Kobayashi, M., Nozawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+transmembrane+potential+of+bacterial+cells+by+voltage-sensitive+dyes.">Probing the transmembrane potential of bacterial cells by voltage-sensitive dyes.</a> Anal. Sci. 19 (9), 1239-1242 (2003).</li><li>Breeuwer, P., Abee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+membrane+potential,+intracellular+pH+and+respiration+of+bacteria+employing+fluorescence+techniques.">Assessment of the membrane potential, intracellular pH and respiration of bacteria employing fluorescence techniques.</a> Mol. Microb. Ecol. Manual. 8, 1563-1580 (2004).</li><li>Mortimer, F. C., Mason, D. J., Gant, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+monitoring+of+antibiotic-induced+injury+in+Escherichia+coli+using+cell-impermeant+fluorescent+probes.">Flow cytometric monitoring of antibiotic-induced injury in Escherichia coli using cell-impermeant fluorescent probes.</a> Antimicrob. Agents Chemother. 44 (3), 676-681 (2000).</li><li>Clementi, E. A., Marks, L. R., Duffey, M. E., Hakansson, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Initiation+Mechanism+of+Death+in+Streptococcus+pneumoniae+Induced+by+the+Human+Milk+Protein-Lipid+Complex+HAMLET+and+Activated+during+Physiological+Death.">A Novel Initiation Mechanism of Death in Streptococcus pneumoniae Induced by the Human Milk Protein-Lipid Complex HAMLET and Activated during Physiological Death.</a> J. Biol. Chem. 287 (32), 27168-27182 (2012).</li><li>Trombe, M. C., Laneelle, G., Sicard, 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=Characterization+of+a+Streptococcus+pneumoniae+mutant+with+altered+electric+transmembrane+potential.">Characterization of a Streptococcus pneumoniae mutant with altered electric transmembrane potential.</a> J. Bacteriol. 158 (3), 1109-1114 (1984).</li><li>Trombe, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+calcium+porter+of+Streptococcus+pneumoniae+involved+in+calcium+regulation+of+growth+and+competence.">Characterization of a calcium porter of Streptococcus pneumoniae involved in calcium regulation of growth and competence.</a> J. Gen. Microbiol. 139 (3), 433-439 (1993).</li><li>Marks, L. R., Clementi, E. A., Hakansson, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitization+of+Staphylococcus+aureus+to+Methicillin+and+Other+Antibiotics+In+Vitro+and+In+Vivo+in+the+Presence+of+HAMLET.">Sensitization of Staphylococcus aureus to Methicillin and Other Antibiotics In Vitro and In Vivo in the Presence of HAMLET.</a> PLoS ONE. 8 (5), (2013).</li><li>Futsaether, C. M., Johnsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fura-2+to+measure+intracellular+free+calcium+in+Propionibacterium+acnes.">Using fura-2 to measure intracellular free calcium in Propionibacterium acnes.</a> Can. J. Microbiol. 40 (6), 439-445 (1994).</li><li>Tisa, L. S., Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+properties+of+Escherichia+coli+mutants+having+abnormal+Ca2++content.">Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content.</a> J. Bacteriol. 177 (24), 7112-7118 (1995).</li><li>Werthen, M., Lundgren, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++mobilization+and+kinase+activity+during+acylated+homoserine+lactone-dependent+quorum+sensing+in+Serratia+liquefaciens.">Intracellular Ca2+ mobilization and kinase activity during acylated homoserine lactone-dependent quorum sensing in Serratia liquefaciens.</a> J. Biol. Chem. 276 (9), 6468-6472 (2001).</li><li>Avery, O. T., MacLeod, C. M., McCarty, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+chemical+nature+of+the+substance+inducing+transformation+of+pneumococcal+types.+Induction+of+transformation+by+a+dexoxyribonuceic+acid+fraction+isolated+from+pneumococcus+type+III.">Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a dexoxyribonuceic acid fraction isolated from pneumococcus type III.</a> J. Exp. Med. 79, 137-158 (1944).</li><li>Clementi, E. A., Wilhelm, K. R., Schleucher, J., Morozova-Roche, L. A., Hakansson, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Complex+of+Equine+Lysozyme+and+Oleic+Acid+with+Bactericidal+Activity+against.">A Complex of Equine Lysozyme and Oleic Acid with Bactericidal Activity against.</a> PLoS One. . 8, (2013).</li></ol>

The authors have no competing financial interests to declare.

Despite the limitation that size presents for using classic electrophysiology methods to detect changes in polarity and integrity of the membrane and changes in ion concentrations within bacteria, we have described a way to measure these events in S. pneumoniae using fluorescent dyes. Our protocols are the first of their kind described for the pneumococcus and one of the few described for bacterial species in general. By using a fluorescence detection plate reader, these events can be measured in small, 200 &#18...

Our lab has identified a protein-lipid complex from human milk named HAMLET (for Human Alpha-lactalbumin Made LEthal to Tumor cells) that induces apoptosis in tumor cells, but is also able to kill a variety of bacterial species1,2. The species that were found to be particularly sensitive were those that target the respiratory tract, with Streptococcus pneumoniae (the pneumococcus) displaying the greatest sensitivity and an apoptosis-like phenotype of death2,3. Membrane depolarization and specific ion transport events are well-described and crucial events during apoptosis in eukaryotic cells, particular in the mitochondria, where radioactive TPP+ ions and fluorescent dyes including JC-1 and TMRE have been used to demonstrate depolarization of the mitochondrial membrane3-5. Thus, we sought to learn more about the effect of HAMLET on these membrane-related features in the pneumococcus as we focused our efforts to develop a better understanding of the mechanistic components of the apoptosis-like phenotype in bacteria, with great potential for identifying novel antibacterial therapies or vaccine candidates in the process.
In seeking to establish protocols for our mechanistic studies, we discovered that in contrast to the well-described methodology in eukaryotic systems, there are very few published studies detailing electrophysiology and ion transport mechanisms of the bacterial membrane6,7. This is primarily attributed to the smaller size of microorganisms and their surface architecture, particularly the presence of cell wall, that restricts accessibility of the membrane for use of conventional eukaryotic methods like patch clamping, although some studies using giant protoplasts have been performed with mixed success8,9. As work with these giant protoplasts is not an ideal or even practical method for most bacterial species since it requires manipulated bacteria in an unnatural and abiotic state, the limited studies of bacterial membrane polarity that have been performed have primarily employed flow cytometry and the use of cyanine and oxonol fluorescent dyes10-16.
Instead of flow cytometry, which gathers individual fluorescence readings from one bacterium at a single time point, we chose to use a fluorescence detection plate reader to detect fluorescence intensity of bacterial suspensions in a 96-well plate format over time. This enabled us to treat a population of bacteria at various time points with much greater simplicity and ease, and continuously monitor the fluorescence kinetics of the entire population for extended periods of time, which is difficult to achieve using flow cytometry. After testing a wide variety of the potential-sensitive fluorescent dyes (including those mentioned above for use with the mitochondria), we achieved the best technical and practical success using the bis-oxonol dye called DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) to monitor changes in polarity.
We also found it valuable to concurrently monitor disruptions in the integrity of the membrane using propidium iodide (PI). This dye fluoresces upon binding to nucleic acid, but is only able to do so when the integrity of the bacterial membrane is compromised, making it the popular component used to detect dead cells in live-dead staining assays. In addition to PI, SYTOX green and TO-PRO-1 are fluorescent dyes that are similar in action and have been previously used for bacteria in a few studies using flow cytometry detection methods17. We chose to use PI due to its excitation wavelength that allowed us to monitor its fluorescence concurrently with DiBAC in a given sample.
In our studies, we have observed that HAMLET, as well as another related protein-lipid complex with bactericidal activity known as ELOA, induced depolarization and rupture of the bacterial membrane as indicated by increases in the fluorescence of both dyes upon treatment of the pneumococci3,18,26. For both complexes, we observed that the fluorescence intensity of DiBAC4(3) increased prior to the increase in intensity of PI, indicating that depolarization occurred prior to rupture and is, therefore, a specific event induced by our bactericidal protein-lipid complexes of interest. This distinction is important to make, as rupture of the membrane can itself cause nonspecific depolarization. Measuring and analyzing both DiBAC4(3) and PI fluorescence kinetics concurrently allowed us to examine this relationship between the two membrane events, which is an additional advantage of using fluorometry instead of flow cytometry.
To monitor bacterial ion flux, there has been some previous success with using radioisotopes, including measuring uptake of 45Ca2+ in the pneumococcus19,20, which we have also used in our recent studies18,21. However, working with these radioactive ions has several drawbacks. They can be expensive, time-consuming, and messy, and can also expose the individual performing the experiment to harm, depending on the isotope of interest. Additionally, it is difficult to monitor rapid changes over time. Thus, we turned towards an alternative method of measurement that employs acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. By itself, the indicator dye is charged and does not pass through the membrane easily, but with the addition of the lipophilic ester group, the now uncharged molecule can pass through the membrane of the bacterium. Upon entering the interior, the bacterial esterases cleave the ester group, leaving the dye free inside the cell and charged again, significantly slowing its ability to exit the cell and allowing the dye to accumulate inside over time. However, use of these ester dyes has only been described in a few bacterial species to detect changes in intracellular Ca2+ 22-24 and H+ 16, with varying methods of loading, detection, and success.
With a desire to monitor changes in intracellular Ca2+ and also K+ and H+ levels in S. pneumoniae upon treatment with HAMLET and other compounds, we successfully created protocols to efficiently load fluorescent indicator dyes into bacterial cells. Effective loading into bacteria required both probenecid that increases dye retention by blocking anion-transporters and PowerLoad, a proprietary compound from Life Technologies that increases loading efficiency. Fura-2/AM (detecting Ca2+), PBFI/AM (detecting K+), and BCECF/AM (detecting H+) were successfully loaded into both unencapsulated and encapsulated pneumococcal strains enabling &#160;measurement of the resulting fluorescence patterns after addition of ionophores, such as ionomycin (Ca2+ uncoupler), valinomycin (K+ uncoupler) and CCCP (H+ uncoupler) using a fluorescence detection plate reader18,21.

<table border="0" cellpadding="0" cellspacing="0" width="864">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Todd Hewitt</td>
			<td style="width:139px;">Bacto, BD Diagnostics</td>
			<td style="width:143px;">249240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Yeast Extract</td>
			<td style="width:139px;">Bacto, BD Diagnostics</td>
			<td style="width:143px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Phosphate Buffered Saline (PBS; pH 7.2)</td>
			<td style="width:139px;">Invitrogen (GIBCO)</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Dimethyl sulfoxide</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">D5879</td>
			<td>DMSO</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:359px;">DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol)</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">B-438</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Propidium Iodide</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4170</td>
			<td>Make up in deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-(+)-Glucose</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PowerLoad</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P10020</td>
			<td>100X concentrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Probenecid</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P36400</td>
			<td>Make 100X stock by adding 1 ml of PBS to one 77 mg vial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Fura-2/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">F1221</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PBFI/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P1267</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Nigericin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">N7143</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">3246-01</td>
			<td>monobasic</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">K2HPO4</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">4012-01</td>
			<td>dibasic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">NaOH</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">5565-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BCECF/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">B1170</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">CCCP</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;"></td>
			<td>Protonophore that causes an influx of H+ into the cytoplasm,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>&#160;dissipating the electrical potential and the H+ gradient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Culture tube&#160;</td>
			<td style="width:139px;">VWR</td>
			<td style="width:143px;">53283-802</td>
			<td>Fits the Spectronic spectrophotometer; borosilicate glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Spectrophotometer</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:143px;"></td>
			<td>Spectronic 20D+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">15 ml plastic conical tube</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Clear 96-well polystyrene microtiter plate</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-565-501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Plate reader</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:143px;">Synergy 2 Multi-Mode&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;"></td>
			<td style="width:139px;"></td>
			<td style="width:143px;">Microplate Reader</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Gen5 software</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:143px;">Gen5&#8482; Software</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in streptococcus pneumoniae using fluorescent dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

For all experiments, there is one sample and set of conditions present in each well. Thus, each tracing represents the fluorescence intensity of an entire population of bacteria over time. The results should be easily interpretable, with a clear distinction between the fluorescence of the treated samples and that of the untreated controls. The kinetics and degree of an observed change in fluorescence could provide information about the possible mechanism and extent of the event being monitored.
<p class="jov...

Watch this Scientific Journal Video about Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes at JoVE.c

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

monitoring-changes-membrane-polarity-membrane-integrity-intracellular

Research

JoVE Journal

Immunology and Infection

13.5K Views.  University at Buffalo, State University of New York. Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Video: Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...