Name: Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization (Video) | JoVE Journal
Uploaded: 2015-07-28T15:00:00.000+00:00
Duration: 9 min 25 s
Description: Learn to quantify cytosolic versus vacuolar Salmonella typhimurium in bone marrow-derived macrophages using differential digitonin permeabilization and FACS analysis. This method reveals pathogen localization dynamics.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We would like to acknowledge Mathias S. Dick, Roland F. Dreier and Sebastian R&#252;hl for discussion. This work was supported by an SNSF Professorship PP00P3_139120/1 and a University of Basel project grant ID2153162 to P.B.

1. Digitonin Assay with mCherry-expressing Salmonella (FACS-based Analysis)
<ol>
	<li>Preparing bacteria 
	Note: Salmonella wild-type SL1344 (Streptomycin resistant) expressing mCherry from a plasmid (pFPV encoding mCherry under the rpsM promoter) is used in this assay. Bacterial phagocytosis and proliferation where not altered by the constitutive expression of mCherry (data not shown)&#160;16.
	<ol>
		<li>Inoculate the strain 1 day before the infection in 3 ml of LB medium supplemented with Streptomycin (90 &#181;g/ml) and Ampicillin (100 &#181;g/ml, mCherry expressing vector) in a shaker at 37 &#176;C O/N.</li>
	</ol>
	</li>
	<li>Plating cells for infection.
	<ol>
		<li>Seed wild-type BMDMs in a 24 well plate at a density of 1.25 x 105 cells/well in 1 ml of primary macrophage medium (DMEM supplemented with 10% Fetal Calf Serum (FCS, heat-inactivated, 56 &#176;C, 30 min), 1% HEPES, 1% Non-essential amino acids (NEAA), 1% L-Glutamine and 10% L929 supernatant (conditioned medium from macrophage colony-stimulating factor (M-CSF) producing L929 cells). Seed wells according to Table 1 to account for the individual conditions. Add coverslips to the wells used as controls (Table 1).</li>
		<li>Incubate the cells overnight in an incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td colspan="2">Permeabilization</td>
			<td colspan="3">Staining</td>
		</tr>
		<tr>
			<td></td>
			<td>Glass coverslip</td>
			<td>Salmonella</td>
			<td>Digitonin</td>
			<td>Saponin</td>
			<td>anti-Salmonella</td>
			<td>anti-Calnexin</td>
			<td>anti-PDI</td>
		</tr>
		<tr>
			<td>A1 (sample)</td>
			<td>-/+*</td>
			<td>+</td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A2 (sample)</td>
			<td>-/+*</td>
			<td>+</td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A3 (sample)</td>
			<td>-/+*</td>
			<td>+</td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A4 (no permeabilization control)</td>
			<td>-/+*</td>
			<td>+</td>
			<td></td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A5 (complete permeabilization control)</td>
			<td>-/+*</td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td>+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B1 (stained for calnexin)</td>
			<td>+</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>+</td>
			<td></td>
		</tr>
		<tr>
			<td>B2 (stained for calnexin)</td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
			<td>+</td>
			<td></td>
		</tr>
		<tr>
			<td>B3 (stained for calnexin)</td>
			<td>+</td>
			<td></td>
			<td></td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td></td>
		</tr>
		<tr>
			<td>C1 (stained for PDI)</td>
			<td>+</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>+</td>
		</tr>
		<tr>
			<td>C2 (stained for PDI)</td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
			<td></td>
			<td>+</td>
		</tr>
		<tr>
			<td>C3 (stained for PDI)</td>
			<td>+</td>
			<td></td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
			<td>+</td>
		</tr>
	</tbody>
</table>
Table 1: Plating scheme for bone-marrow derived macrophages (BMDMs) in a 24-well format for subsequent infection with Salmonella, permeabilization, and staining. *Depending on whether protocol 1 or protocol 2 is done.
<ol start="3">
	<li>Preparing the bacteria for infection.
	<ol>
		<li>The next day, determine the concentration of Salmonella in the overnight culture by diluting the culture 1:10 in PBS and measuring the OD600 against a PBS only control. This ensures that the OD600 is measured in the linear range of the spectrophotometer.</li>
		<li>Calculate the concentration of Salmonella per ml. An OD600 of 1 corresponds to 1 x 109 bacteria.</li>
		<li>Dilute the bacteria in macrophage medium to reach a Salmonella concentration of 1.25 x 106 cells/ml.</li>
	</ol>
	</li>
	<li>Infection of cells with Salmonella.
	<ol>
		<li>Remove medium from BMDMs. Add 1 ml of macrophage medium to uninfected control wells (B1-B3, C1-C3). Add 1 ml of Salmonella suspension to wells A1 - A5 to reach a multiplicity of infection (m.o.i.) of 10 bacteria per cell.</li>
		<li>Centrifuge the plate for 15 min at 300 x g at 37 &#176;C to synchronize the infection. Transfer the plate in an incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Killing extracellular Salmonella with Gentamicin.
	<ol>
		<li>At 1 hr post-infection, remove plate from incubator and add 0.1 ml of macrophage medium containing 0.1 mg/ml&#160;Gentamicin to kill extracellular bacteria. Add Gentamicin to all wells, even to uninfected controls, to ensure that all cells are treated the same way. Transfer the plate in an incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Washing the cells.
	<ol>
		<li>At 2 hr&#160;post infection, remove plate from incubator and wash all wells 2x with 1 ml of plain DMEM (pre-warmed to 37 &#176;C) and replace it with macrophage medium (pre-warmed to 37 &#176;C) containing 10 &#181;g/ml&#160;Gentamycin to prevent growth of any remaining extracellular bacteria. Transfer the plate in an incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Preparing fresh buffers with detergents and antibodies.
	<ol>
		<li>Just before the desired time point of analysis, prepare a fresh stock solution of digitonin at 1 mg/ml&#160;in KHM Buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl2, pH 7.3). Filter the stock and dilute in KHM buffer to a working concentration of 50 &#181;g/ml&#160;of digitonin. In addition, prepare a solution of 0.1% Saponin in KHM Buffer, anti-Salmonella antibody coupled to FITC (CSA-1-FITC) at 1/500, anti-calnexin at 1/100 or anti-PDI at 1/100 in KHM Buffer supplemented with 3% BSA.</li>
	</ol>
	</li>
	<li>Cell permeabilization.
	<ol>
		<li>Remove plate from incubator and wash wells 3x&#160;with 0.5 ml of KHM Buffer (pre-warmed to 37 &#176;C). Remove KHM Buffer and add 0.25 ml plain KHM Buffer to wells A4 and B1/C1, KHM Buffer with digitonin to wells A1 - A3 and B2/C2 or KHM Buffer with saponin to wells A5 and B3/C3 (according to Table 1). Incubate for exactly 1 min at RT and immediately wash all wells 3x with 0.5 ml of KHM Buffer (pre-warmed to 37 &#176;C).</li>
	</ol>
	</li>
	<li>Primary antibody staining.
	<ol>
		<li>Remove the KHM Buffer and add the primary antibody solutions (Goat anti Salmonella-FITC, 250 &#181;l/well, 0.1 &#181;g/ml) to the appropriate wells (Figure 1, Table 1). Incubate the plate for 15 min in an incubator at 37 &#176;C and 5% CO2.</li>
		<li>Wash the cells 1x with 1 mL PBS.</li>
	</ol>
	</li>
	<li>Infected Cells (wells A1 - A5): FACS analysis
	<ol>
		<li>Wash cells 3x with PBS and add 0.5 mL of ice-cold PBS containing 0.1% Triton. Transfer to a 5 mL FACS tube with cell strainer cap to break up cell aggregates. Keep samples on ice.</li>
		<li>Analyze the samples of a FACS. Using the fully permeabilized controls, set the gate for total bacteria based on forward and side scatter (FSC/SSC) first, and the mCherry signal (610 nm &#177; 20 nm filter).</li>
		<li>Next, analyze the FITC signal in the total bacterial population using the fully permeabilized and not permeabilized controls to set the gates for cytosolic bacteria (FITC+, mCherry+) and vacuolar bacteria (FITC-, mCherry+) (Figure 3).</li>
		<li>With the gates set, analyze the samples and determine the percentage of cytosolic vs. vacuolar bacteria using FlowJo software according to manufacturer&#8217;s protocol.</li>
	</ol>
	</li>
	<li>Uninfected permeabilization controls (wells B1 - B3, C1 - C3): microscopy
	<ol>
		<li>Fixation
		<ol>
			<li>Remove PBS and add 250 &#181;l of PFA 4% in PBS. Incubate for 10 min at 37 &#176;C.</li>
			<li>Wash wells 2x with 1 ml of PBS and add 250 &#181;l of 0.1 M glycine in PBS for 10 min to quench the fixative. Wash wells 2x with 1 ml of PBS.</li>
		</ol>
		</li>
		<li>Secondary antibody staining.
		<ol>
			<li>Stain permeabilization controls for 1 hr at RT with appropriate secondary antibodies coupled to fluorophores in PBS supplemented with 0.1% Saponin and 3% BSA to fully permeabilize cells.</li>
			<li>Wash coverslips 3x with 1 ml of PBS and mount the coverslips for analysis in mounting medium with DAPI (1.5 &#956;g/ml).</li>
		</ol>
		</li>
		<li>Microscopy analysis.
		<ol>
			<li>Analyze the coverslips with the controls at a 40X or 63X magnification using a confocal fluorescence microscope. If the permeabilization has worked, there should be no staining for non-permeabilized cells, Calnexin staining for both digitonin- and Saponin-permeabilized cells and PDI staining only for saponin-permeabilized cells (Figure 4).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>



2. Digitonin Assay with Unlabeled Salmonella (Microscopy-based Analysis)



<ol>
	<li>Preparing bacteria for O/N cultures.
	<ol>
		<li>Inoculate unlabeled Salmonella wild-type SL1344 (Streptomycin resistant) 1 day before the infection in 3 ml of LB medium supplemented with Streptomycin (90 &#181;g/ml) in a shaker at 37 &#176;C O/N.</li>
		<li>Plating cells for infection.
		<ol>
			<li>Seed BMDMs in a 24 well plate containing sterile glass coverslips at a density of 1.25 x 105 cells/well in 1 ml of macrophage medium (DMEM supplemented with 10% Fetal Calf Serum (FCS, de-complemented, 56 &#176;C, 30 min), 1% HEPES, 1% L-Non-essential amino acids (NEAA), 1% L-Glutamine and 10% L929 supernatant). Seed wells according to Table 1 to account for the individual conditions.</li>
			<li>Incubate the cells O/N in an incubator at 37 &#176;C and 5% CO2.</li>
		</ol>
		</li>
		<li>Preparing the bacteria for infection.
		<ol>
			<li>The next day, determine the concentration of Salmonella in the O/N culture by diluting the culture 1:10 in PBS and measuring the OD600 against a PBS only control. This ensures that the OD600 is measured in the linear range of the spectrophotometer.</li>
			<li>Determine the concentration of Salmonella per milliliter. An OD600 of 1 corresponds to 1 x 109 bacteria.</li>
			<li>Dilute the bacteria in macrophage medium to reach a Salmonella concentration of 1.25 x 106&#160;cells/ml.</li>
		</ol>
		</li>
		<li>Infection of cells with Salmonella.
		<ol>
			<li>Remove medium from all wells. Add 1 ml of plain macrophage medium to uninfected control wells (B1 - B3, C1 - C3). Add 1 ml of Salmonella suspension to wells A1 - A5 to reach a multiplicity of infection (m.o.i.) of 10 bacteria per cell.</li>
			<li>Centrifuge the plate for 15 min at 300 x g at 37 &#176;C to synchronize the infection. Transfer the plate in an incubator at 37 &#176;C and 5% CO2.</li>
		</ol>
		</li>
		<li>Killing extracellular Salmonella with Gentamycin.
		<ol>
			<li>At 1 hr post-infection, remove plate from incubator and add 0.1 ml of macrophage medium containing 1 mg/ml Gentamycin to kill extracellular bacteria to all wells. Transfer the plate in an incubator at 37 &#176;C and 5% CO2.</li>
		</ol>
		</li>
		<li>Washing the cells.&#160;
		<ol>
			<li>At 2 hr&#160;post-infection, remove plate from incubator and wash each well 3x with 1 ml of fresh macrophage medium containing 10 &#181;g/ml Gentamycin to prevent growth of any remaining extracellular bacteria. Transfer the plate in an incubator at 37&#176;C and 5% CO2.</li>
		</ol>
		</li>
		<li>Preparing fresh buffers with detergents and antibodies.&#160;
		<ol>
			<li>Just before the desired time point of analysis, prepare a fresh stock solution of digitonin at 1 mg/ml in KHM Buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl2, pH 7.3). Filter the stock and dilute in KHM Buffer to a working concentration of 50 &#181;g/ml of digitonin.</li>
			<li>In addition, prepare a solution of 0.1% Saponin in KHM Buffer, anti-Salmonella-FITC (CSA-1, 0.1 &#181;g/ml), anti-calnexin at 1/100 or anti-PDI at 1/100 in KHM Buffer supplemented with 3% BSA (see materials table for antibody concentration).</li>
		</ol>
		</li>
		<li>Cell permeabilization.
		<ol>
			<li>Remove plate from incubator and wash wells 3x with 0.5 ml of KHM Buffer (pre-warmed to 37 &#176;C). Remove KHM Buffer and add 0.25 ml plain KHM Buffer to wells A4 and B1/C1, KHM Buffer with digitonin to wells A1 - A3 and B2/C2 or KHM Buffer with saponin to wells A5 and B3/C3 (according to Table 1). Incubate for exactly 1 min at RT and immediately wash all wells 3x with 0.5 ml of KHM Buffer (pre-warmed to 37 &#176;C).</li>
		</ol>
		</li>
		<li>Primary antibody staining.
		<ol>
			<li>Remove the KHM Buffer and add the primary antibody solutions (Goat anti Salmonella CSA1-FITC, 250 &#181;l/well, 0.1 &#181;g/ml) to the appropriate wells (Figure 2, Table 1). Incubate the plate for 15 min in an incubator at 37 &#176;C and 5% CO2. Wash 3x with 1 ml PBS.</li>
		</ol>
		</li>
		<li>Fixation.
		<ol>
			<li>Remove PBS and add 250 &#181;l of PFA 4% in PBS. Incubate for 10 min at 37 &#176;C.</li>
			<li>Wash wells 2x with 1 ml of PBS and add 250 &#181;l of 0.1 M Glycine in PBS for 10 min to quench the fixative. Wash wells 2x with 1 ml of PBS.</li>
		</ol>
		</li>
		<li>Total Salmonella staining of infected samples.
		<ol>
			<li>Wash coverslips 3x with PBS with 0.1% Saponin/3% BSA and incubate the samples with an anti-CSA1 antibody (Goat anti-CSA1, 0.1 &#181;g/ml, mouse anti-LPS works as well, 0.1 &#181;g/ml) for 1 hr at RT in PBS with 0.1% Saponin/3% BSA.</li>
			<li>Wash 3x in PBS with 0.1% Saponin/3% BSA. Incubate your samples with a secondary anti-Goat antibody coupled to fluorophore of choice in PBS with 0.1% Saponin/3% BSA for 30 min at RT in the dark.</li>
			<li>Wash coverslips 3x with 1 ml of PBS and mount the coverslips for analysis in mounting medium with DAPI (1.5 &#956;g/ml).&#160;&#160;</li>
		</ol>
		</li>
		<li>Microscopy analysis.
		<ol>
			<li>Acquire data with a confocal microscope by counting cytosolic Salmonella (anti-Salmonella-FITC positive) and total Salmonella (double positive) (Figure 5). Permeabilization controls should show: no staining for non-permeabilized cells, Calnexin staining for digitonin-permeabilized cells and PDI staining for saponin-permeabilized cells (Figure 4). Wells A4 and A5 will serve as internal controls for the Salmonella staining.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Kumar, Y., Valdivia, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leading+a+sheltered+life%3A+intracellular+pathogens+and+maintenance+of+vacuolar+compartments%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">Leading a sheltered life: intracellular pathogens and maintenance of vacuolar compartments.</a> Cell Host Microbe. 5, 593-601 (2009).</li>
<li>Chong, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+early+phagosomal+stage+of+Francisella+tularensis+determines+optimal+phagosomal+escape+and+Francisella+pathogenicity+island+protein+expression%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression.</a> Infect Immun. 76, 5488-5499 (2008).</li>
<li>Mellouk, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shigella+subverts+the+host+recycling+compartment+to+rupture+its+vacuole%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">Shigella subverts the host recycling compartment to rupture its vacuole.</a> Cell Host Microbe. 16, 517-530 (2014).</li>
<li>Checroun, C., Wehrly, T. D., Fischer, E. R., Hayes, S. F., Celli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy-mediated+reentry+of+Francisella+tularensis+into+the+endocytic+compartment+after+cytoplasmic+replication%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication.</a> Proc Natl Acad Sci U S A. 103, 14578-14583 (2006).</li>
<li>Broz, P., Monack, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Newly+described+pattern+recognition+receptors+team+up+against+intracellular+pathogens%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">Newly described pattern recognition receptors team up against intracellular pathogens.</a> Nat Rev Immunol. 13, 551-565 (2013).</li>
<li>Hagar, J. A., Powell, D. A., Aachoui, Y., Ernst, R. K., Miao, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytoplasmic+LPS+activates+caspase-11%3A+implications+in+TLR4-independent+endotoxic+shock%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock.</a> Science. 341, 1250-1253 (2013).</li>
<li>Ogawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Escape+of+intracellular+Shigella+from+autophagy%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Escape of intracellular Shigella from autophagy.</a> Science. 307, 727-731 (2005).</li>
<li>Haraga, A., Ohlson, M. B., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salmonellae+interplay+with+host+cells%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Salmonellae interplay with host cells.</a> Nat Rev Microbiol. 6, 53-66 (2008).</li>
<li>Birmingham, C. L., Smith, A. C., Bakowski, M. A., Yoshimori, T., Brumell, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy+controls+Salmonella+infection+in+response+to+damage+to+the+Salmonella-containing+vacuole%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole.</a> J Biol Chem. 281, 11374-11383 (2006).</li>
<li>Malik-Kale, P., Winfree, S., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+bimodal+lifestyle+of+intracellular+Salmonella+in+epithelial+cells%3A+replication+in+the+cytosol+obscures+defects+in+vacuolar+replication%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The bimodal lifestyle of intracellular Salmonella in epithelial cells: replication in the cytosol obscures defects in vacuolar replication.</a> PLoS One. 7, e38732(2012).</li>
<li>Zhao, Y. O., Khaminets, A., Hunn, J. P., Howard, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+the+Toxoplasma+gondii+parasitophorous+vacuole+by+IFNgamma-inducible+immunity-related+GTPases+%28IRG+proteins%29+triggers+necrotic+cell+death%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death.</a> PLoS Pathog. 5, e1000288(2009).</li>
<li>Meunier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspase-11+activation+requires+lysis+of+pathogen-containing+vacuoles+by+IFN-induced+GTPases%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.</a> Nature. 509, 366-370 (2014).</li>
<li>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cluster+of+interferon-gamma-inducible+p65+GTPases+plays+a+critical+role+in+host+defense+against+Toxoplasma+gondii%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">A cluster of interferon-gamma-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.</a> Immunity. 37, 302-313 (2012).</li>
<li>Degrandi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+guanylate+binding+protein+2+%28mGBP2%29+controls+Toxoplasma+gondii+replication%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication.</a> Proc Natl Acad Sci U S A. 110, 294-299 (2013).</li>
<li>Mariathasan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+activation+of+the+inflammasome+by+caspase-1+adaptors+ASC+and+Ipaf%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.</a> Nature. 430, 213-218 (2004).</li>
<li>Knodler, L. A., Nair, V., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+assessment+of+cytosolic+Salmonella+in+epithelial+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Quantitative assessment of cytosolic Salmonella in epithelial cells.</a> PLoS One. 9, e84681(2014).</li>
<li>Ng, H., Smith, D. J., Nagley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+flow+cytometry+to+determine+differential+redistribution+of+cytochrome+c+and+Smac%2FDIABLO+from+mitochondria+during+cell+death+signaling%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.</a> PLoS One. 7, e42298(2012).</li>
<li>Krawczyk, E., Suprynowicz, F. A., Sudarshan, S. R., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+orientation+of+the+human+papillomavirus+type+16+E5+oncoprotein%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Membrane orientation of the human papillomavirus type 16 E5 oncoprotein.</a> J Virol. 84, 1696-1703 (2010).</li>
<li>Keller, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+measurements+of+vacuolar+rupture+caused+by+intracellular+pathogens%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Single cell measurements of vacuolar rupture caused by intracellular pathogens.</a> J Vis Exp. , e50116(2013).</li>
</ol>

We have nothing to disclose.

Differential permeabilization is an easy and robust method to analyze and quantify the subcellular distribution of bacterial pathogens between vacuolar compartments and the cytosol. The same assay has been used successfully with bacteria such as Francisella novicida2,4 and Shigella flexneri12. However, since many intracellular pathogen alter or modify the host endomembrane structures, the robustness of the digitonin permeabilization will have to be determined on an individual basis...

Intracellular bacterial pathogens replicate either directly in the host cell cytosol, or in specialized vacuolar compartments1. During the initial stage of the infection most pathogens get internalized either by phagocytosis by specialized cells (such as macrophages) or by actively promoting their own uptake into non-phagocytic cells. In phagocytic cells, the phagosome normally fuses with lysosomes to form a degradative compartment, where the phagocytosed particles are digested. Specialized cytosolic bacteria, such as Francisella novicida or Shigella flexneri, escape phagosomal degradation by inducing the rupture of the phagosome and subsequently escape into the cytosol2,3. This requires virulence-associated mechanism such as the Francisella Pathogenicity Island (FPI) or the Shigella flexneri T3SS, which injects effector proteins that promote vacuolar rupture2-4. The host cell cytosol features a number of conserved pattern recognition receptors that normally recognize the presence of pathogens and induce innate immune signaling5. In addition, xenophagy, an anti-microbial form of autophagy can degrade bacteria that enter the cytosol. Cytosolic bacteria are normally well adapted to blunt or circumvent these responses using different strategies. For example, Francisella modifies its LPS to avoid host recognition and Shigella prevents autophagy recruitment using secreted effector proteins6,7.
Another strategy to escape lysosomal degradation is employed by the model vacuolar pathogen Salmonella enterica serovar Typhimurium, thereafter referred to as Salmonella typhimurium. Salmonella uses a T3SS to inject effectors that remodel the initial phagosome into its intracellular niche, the Salmonella-containing vacuole (SCV) 8,9. Continuous manipulation of host pathways is necessary to maintain this vacuole by recruiting lipids and other nutrients to the vacuole. Indeed, a sifA mutant of Salmonella fails to maintain vacuolar stability, and enters the cytosol of host cells within hours after infection, which results in activation of innate immune pathways and autophagy recruitment8. Vacuolar escape of Salmonella varies depending on the cell type that is studies, and several reports have shown that in epithelial cells even WT Salmonella can escape the SCV and hyperreplicate in the cytosol10. Recent reports show that innate immune detection of vacuolar pathogens also depends on the ability of the host to recognize and destabilize pathogen-containing vacuoles (PCVs) 11-14.
Given that both the host or bacteria genotype can affect the distribution of intracellular bacteria between the vacuolar and cytosolic compartment, it is necessary to quantify the number of vacuolar vs. cytosolic bacteria. Since, in most experimental setups host cells are infected with more than one bacterium, and subsequently can harbor several bacteria in different subcellular compartments, a technique is needed that allows quantification on the level of single bacteria. Differential permeabilization (also known as phagosomal protection assay) provides this resolution 2,4,10,12. The assay is based on selective permeabilization of the host cell plasma membrane with the detergent digitonin, which leaves vacuolar membranes intact and thus allows selective staining of cytosolic bacteria with antibodies. Here we provide two protocols for the quantification of cytosolic and vacuolar Salmonella typhimurium using differential permeabilization. The principle of this method is described in Figure 1: Bone-marrow derived macrophages (BMDMs) are infected with stationary phase mCherry-expressing Salmonella. Stationary phase Salmonella need to be used because they downregulate the expression of the SPI-1 T3SS, whose activity would otherwise be recognized by the NLRC4 inflammasome and induce rapid cell death (pyroptosis) of the BMDMs15. Following the infection cells are washed and treated with 50 &#956;g/ml&#160;of digitonin for 1 minute. Cells are washed again immediately and incubated with anti-Salmonella antibodies coupled to FITC to mark bacteria with access to the cytosol. After an additional washing step cells are lysed and the percentage of mCherry+ (vacuolar) and FITC+ / mCherry+ (cytosolic) bacteria is determined by FACS. We also report an adaptation of this method that can be used if no fluorescent protein-expressing strains are available (Figure 2). Additional steps are introduced after the FITC labeling in which cells are fixed and completely permeabilized. Thereafter all bacteria are stained with anti-Salmonella antibodies and corresponding secondary antibodies. Detection is then done by microscopy instead of FACS analysis.

<table><tbody><tr><td>Digitonin</td><td>Sigma</td><td>D5628</td><td>50 &amp;mu;g/ml</td></tr><tr><td>PFA</td><td>mpbio</td><td>219998380</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630</td><td></td></tr><tr><td>Potassium Acetate</td><td>Sigma</td><td>791733</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>anti-Salmonella CSA-1-FITC</td><td>KPL</td><td>01-91-99-MG</td><td>1/500</td></tr><tr><td>anti-Salmonella CSA-1</td><td>KPL</td><td>02-91-99-MG</td><td>1/500</td></tr><tr><td>anti-Calnexin</td><td>Enzo Lifesciences</td><td>SPA-860D</td><td>1/100</td></tr><tr><td>anti-PDI</td><td>Enzo Lifesciences</td><td>SPA-890</td><td>1/100</td></tr><tr><td>Saponin</td><td>Sigma</td><td>47036</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vectorlabs</td><td>H-1200</td><td></td></tr><tr><td>Anti Rabbit antibody-488</td><td>Molecular Probes</td><td>A-11070</td><td>1/500</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G8898</td><td></td></tr><tr><td>Gentamicin</td><td>Life Technologies</td><td>15710-49</td><td>100 &amp;mu;g/ml and 10 &amp;mu;g/ml</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5141</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-019</td><td></td></tr><tr><td>DMEM</td><td>Sigma</td><td>D6429</td><td></td></tr><tr><td>NEAA</td><td>Amimed</td><td>5-13K00-H</td><td></td></tr><tr><td>LSM700 Confocal microscope</td><td>Zeiss</td><td></td><td>imaging done at 63X</td></tr><tr><td>FACS Fortessa</td><td>BD technologies</td><td></td><td>detection mCherry 610 nm</td></tr><tr><td>FACS Fortessa</td><td>BD technologies</td><td></td><td>detection FITC 530 nm</td></tr></tbody></table>

quantification of cytosolic vs. vacuolar salmonella in primary macrophages by differential permeabilization

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Figure 1 and Figure 2 show the schematics of the digitonin assay described in protocol 1 and protocol 2 illustrating the critical steps in the protocol and the results obtained. Figure 3 shows typical FACS results. Positive and negative controls are used to set the gates for mCherry+/FITC- bacteria (vacuolar Salmonella) and mCherry+/FITC+ bacteria (cytosolic Salmonella). Based on these gates the percentage of cytosolic and vacuolar bacteria can be determined in the experime...

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

The overall goal of the following experiment is to determine the percentage of cytosolic and vacuolar salmonella tym in infected bone marrow derived macrophages. This is achieved by infecting macrophages with M cherry expressing salmonella Ty aerium. As a second step, cells are treated with the detergent tonin, which permeables the plasma membrane, but not vacuolar membranes.Next cells are stained with an antis salmonella antibody coupled to the Fluor ofor Zi.In order to label cytosolic salmonella ty erum, the results show the percentage of cytosolic bacteria among total bacteria based on fluorescence activated cell sorting or facts. These metals can help answer key question in the field of pathogen interaction, such as does an intracellular pathogen replicate in a VA or in the cytosol Inoculate the bacterial strain one day before the infection In three milliliters of Luria britani medium supplemented with streptomycin and ampicillin in a shaker at 37 degrees Celsius overnight to account for the individual conditions. Add cover slips to the wells of a 24 well plate that will be used as controls to plate the cells for infection seed, wild type bone marrow derived macrophages, or BM dms at a density of 0.125 million cells per well in one milliliter of primary macrophage medium, then incubate the cells overnight in an incubator at 37 degrees Celsius and 5%carbon dioxide.The next day. Determine the concentration of salmonella in the overnight culture by measuring the optical density at 600 nanometers or OD 600 of the diluted culture against phosphate buffered saline. This ensures that the OD 600 is measured in the linear range of the spectrophotometer.Calculate the concentration of salmonella per milliliter and OD 600 of one corresponds to 1 billion. Bacteria dilute the bacteria in macrophage medium to reach a salmonella concentration of 1.25 million cells per milliliter to infect the cells with salmonella. Remove the medium from the B MDMs.Add one milliliter of macrophage medium to the uninfected control wells, B one through B three and C one through C3.Then add one milliliter of salmonella suspension to wells, A one through a five to reach a multiplicity of infection of 10 bacteria per cell. Centrifuge the plate for 15 minutes at 300 times G and 37 degrees Celsius to synchronize the infection before transferring the plate to incubate a 37 degrees Celsius and 5%carbon dioxide at one hour post-infection. Remove the plate from the incubator to add 0.1 milliliter of macrophage medium containing Gentamycin to kill extracellular bacteria.Add gentamycin to all the wells, even the uninfected controls to ensure that all cells are treated the same way. Then transfer the plate to an incubator at 37 degrees Celsius and 5%carbon dioxide at two hours post infection. Remove the plate from the incubator and wash all wells two times with one milliliter of prewarm docos, modified eagles medium or DMEM.Replace the DMEM with prewarm macrophage medium containing gentamicin to prevent growth of any remaining extracellular bacteria before transferring the plate back to the incubator just before the desired time point of analysis. Prepare fresh solutions of KHM buffer KHM buffer with digit toin and KHM buffer with saponin. In addition to antibody solutions as described in the text protocol to perform cell permeation, remove the plate from the incubator and wash the wells three times with 0.5 milliliters of prewarm KHM buffer.Then remove the KHM buffer and add 0.25 milliliters of plain KHM buffer to wells A four B one and C one khm buffer with dig toin to wells, A one through A three B two and C two and khm buffer with saponin to wells A five B three and C3 incubate for exactly one minute at room temperature before immediately washing all wells three times with 0.5 milliliters of PREWARM KHM buffer. Finally, remove the KHM buffer and add the primary antibody solutions to the appropriate wells before incubating the plate for 15 minutes at 37 degrees Celsius and 5%carbon dioxide following incubation. Wash the cells once with one milliliter of PBS for the infected cells in wells, A one through a five.Prepare for fax analysis by washing the cells three times with PBS. Then add 0.5 milliliters of ice cold PBS containing 0.1%Triton. Next, transfer the cell suspension to a five milliliter fax tube with a cell strainer cap to break up cell aggregates while keeping samples on ice.Analyze the samples on a fax machine using the fully permeable controls. Set the gate for total bacteria based on forward and side scatter and the mCherry signal. Next, analyze the fitz signal in the total bacterial population using the fully perme and not perme controls to set the gates for cytosolic bacteria and vacuolar bacteria with the gate set.Analyze the samples and determine the percentage of cytosolic versus vacuolar bacteria using FlowJo software. According to the manufacturer's protocol for the uninfected permeation controls in wells B one through B three and C one through C3, prepare for microscopy by removing the PBS and adding 250 microliters of 4%Paraform aldehyde in PBS incubate for 10 minutes at 37 degrees Celsius following incubation. Wash the wells two times with one milliliter of PBS and at 250 microliters of 0.1 molar glycine in PBS for 10 minutes to quench the fixative, then wash the wells two times with one milliliter of PBS to perform secondary antibody staining.Stain the permeation controls for one hour at room temperature with appropriate secondary antibodies coupled to fluoro fours in PBS. That is supplemented with 0.1%saponin and 3%BSA. To fully perme the cells wash the cover slips three times with one milliliter of PBS and mount the cover slips for analysis in mounting medium with dappy.Analyze the cover slips with the controls at a 40 x or 63 x magnification using a confocal fluorescence microscope. Shown here are typical facts. Results positive and negative controls are used to set the gates for m cherry positive, Fitz positive bacteria, which is cytosolic, salmonella, and m cherry positive FITSI negative, which is valar salmonella.Based on these gates, the percentage of cytosolic and vacuolar bacteria is determined in the experimental samples. In this example, m cherry positive salmonella typhimurium wild type is compared to a CA deletion mutant in bone marrow derived MI macrophages shown here is kexin and PD one staining that is obtained in permeation controls treated with digit, toin or saponin since calnexin is an er membrane protein calnexin staining can be seen throughout the cytosol and around the nucleus in both digit toin and saponin perme cells. In digit toin perme controls no PD one staining is visible.While staining can be observed in sasin and perme cells. An anti salmonella staining result in cells that have been perme with tonin is shown here. Total bacteria are in red while cytosolic bacteria are in green.The E positive population is salmonella with access to the cytosol. While the fite negative population is bacteria that we're residing in an intact salmonella containing vacuole or SCV and we're therefore protected from salmonella FSE labeling here, wild type salmonella is compared to a CFE deletion mutant strain. Since CA is necessary for salmonella to maintain SCV integrity, an increased percentage of salmonella is After watching this video, you should have a good understanding of how to determine the percentage of vascular and cytosolic bacteria.

quantification-cytosolic-vs-vacuolar-salmonella-primary-macrophages

Research

JoVE Journal

Immunology and Infection

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

This video demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the...

An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin,...

An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Learn to quantify cytosolic versus vacuolar Salmonella typhimurium in bone marrow-derived macrophages using differential digitonin permeabilization and FACS analysis. This method reveals pathogen localization dynamics.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Explore More Videos