This work was supported by Innovative Young Biotechnologist Award BT/BI/12/040/2005 and BT/PR13289/BRB/10/745/2009 grant from Department of Biotechnology, Government of India and core funds of Centre for DNA Fingerprinting and Diagnostics, Hyderabad. MNR and GB are the recipients of Junior and Senior Research Fellowships of the Council of Scientific and Industrial Research towards the pursuit of a PhD degree of the Manipal University. SB is the recipient of Junior and Senior Research Fellowship of the Department of Biotechnology towards the pursuit of a PhD degree of the Manipal University.

It is recommended to perform C. glabrata infection experiments in a laboratory with biosafety containment level 2 (BSL-2).
<ol>
	<li>Preparation of THP-1 macrophage monolayer.</li>
	<li>Preparation of C. glabrata cell suspension.</li>
	<li>Infection of THP-1 macrophages with C. glabrata cells.</li>
	<li>Measurement of phagocytosis rate and intracellular replication via colony forming unit assay.</li>
	<li>Monitoring of intracellular replication using confocal laser scanning microscopy.</li>
</ol>
1. Preparation of THP-1 Macrophage Monolayer
<ol>
	<li>THP-1 is a human monocytic cell line derived from the peripheral blood of a 1-year-old male child suffering from acute monocytic leukemia14. THP-1, a suspension cell line, is routinely maintained at 37 &#176;C in humidified 5% CO2 in RPMI-1640 culture medium supplemented with 10% FBS (fetal bovine serum). Treatment with phorbol 12-myristate 13-acetate (PMA, a phorbol ester) results in terminal differentiation of THP-1 monocytic cells into macrophages. Importantly, PMA treatment does not affect cell viability and differentiated cells do not divide.</li>
	<li>Seed approximately 1.5 x&#160;106 THP-1 monocytes in 100 mm culture dishes in RPMI-1640 complete medium (RPMI-1640 medium supplemented with 10% serum, 2 mM glutamine and 1x&#160;antibiotics; 10 ml/dish) and let cells grow at 37 &#176;C in 5% CO2 for 2 days.</li>
	<li>Once THP-1 cells have reached a density of 8 x 105 cells/ml, transfer the culture dish to the cell culture laminar flow hood and gently swirl it to resuspend settled cells.</li>
	<li>Pipette the cell suspension out, place into a 15 ml&#160;sterile tube and centrifuge at 1,000 rpm (130 x g) for 4 min. Discard supernatant and suspend THP-1 cell pellet in 5 ml&#160;fresh, prewarmed, RPMI-1640 complete medium.</li>
	<li>Enumerate cells with a hemocytometer and dilute the cell suspension to a final density of 106 cells/ml&#160;with prewarmed RPMI-1640 complete medium</li>
	<li>Add 1 &#181;l of 160 mM PMA stock (phorbol 12-myristate 13-acetate solution prepared in dimethyl sulfoxide) to 10 ml&#160;THP-1 cell suspension (final concentration 16 nM) and mix well. Dispense 1 ml&#160;cell suspension into each well of a 24-well tissue culture plate and incubate plate in the incubator maintained at 37 &#176;C with 5% CO2 for 12 hr.</li>
	<li>Remove old medium, add 1 ml&#160;prewarmed RPMI-1640 complete medium and allow cells to recover for 12 hr at 37&#176;C in 5% CO2.</li>
	<li>Observe cells under an inverted microscope to ensure that oval-shaped THP-1 monocytic cells in suspension have been differentiated into flattened, spindle-shaped and adherent &#160;macrophages. These differentiated cells are now ready to conduct C. glabrata infection studies.</li>
</ol>
2. Preparation of C. glabrata&#160;Cell Suspension
C. glabrata wild-type strain Bg2 will be used to infect THP-1 macrophages. C. glabrata cells are routinely cultured in liquid YPD (Yeast extract (1%)-Peptone (2%)-Dextrose (2%)) medium. Solid YPD medium is prepared by adding 2% agar before medium autoclaving.
<ol>
	<li>To prepare a culture of C. glabrata, pick a single colony up from the agar plate with a sterile, disposable loop and inoculate into 10 ml&#160;YPD&#160; liquid medium and incubate for 14-16 hr with shaking (200 rpm) at 30 &#176;C.</li>
	<li>Centrifuge this culture (1 ml) at 4,000 rpm (1,800 x g) for 5 min in a table top microcentrifuge, wash cells thrice with sterile PBS (phosphate-buffered saline, pH 7.4) and suspend cell pellet in 1 ml&#160;PBS.</li>
	<li>Measure OD600 of C. glabrata cell suspension and dilute with an appropriate volume of sterile PBS to get a density of 2 x 106 cells/ml&#160;(OD600 = 0.1). Alternatively, the desired cell density can be achieved by counting yeast cells using a hemocytometer.</li>
</ol>
3. Infection of THP-1 Macrophages with C. glabrata&#160;Cells
<ol>
	<li>Add 50 &#181;l C. glabrata cell suspension to THP-1 macrophages (106 cells seeded per well of a 24-well culture plate) to obtain a MOI (multiplicity of infection) of 0.1 and incubate plate at 37 &#176;C with 5% CO2.</li>
	<li>To determine viable yeast counts, dilute 50 &#956;l C. glabrata cell suspension 100-fold in sterile PBS and plate 100 &#956;l on YPD solid medium. Incubate plates at 30 &#176;C and count manually the number of yeast colonies appearing after 1-2 days. These numbers will, henceforth, be referred as 0 hr&#160;C. glabrata CFUs (colony forming units).</li>
	<li>After 2 hr coculturing of THP-1 cells with C. glabrata cells, discard culture medium by inverting 24-well tissue culture plate in a reservoir and wash gently thrice with prewarmed sterile PBS to remove nonphagocytosed extracellular C. glabrata cells. Gentle washes with PBS are absolutely essential to accomplish complete removal of all extracellular yeast without causing any damage to the THP-1 macrophage monolayer.</li>
</ol>
4. Measurement of Phagocytosis Rate and Intracellular Replication via Colony Forming Unit Assay

<ol>
	<li>Lyse C. glabrata infected THP-1 macrophages in 1 ml&#160;sterile H2O for 2 min, scrape gently to remove the macrophage debris from the well and collect cell lysate in a microcentrifuge tube. Microscopic verification of complete macrophage lysis is pivotal for the recovery of all internalized yeast.</li>
	<li>Prepare a 100-fold dilution of lysate in sterile PBS, plate 100 &#956;l on YPD solid medium and incubate plates at 30 &#176;C.</li>
	<li>After 1-2 days, count manually the number of C. glabrata colonies that appeared on YPD medium, multiply with dilution factor (2 hr CFUs) and calculate phagocytosis rate which refers to the percent of C. glabrata cells that are ingested by THP-1 macrophages after 2 hr coincubation &#160;using the following formula. 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;% phagocytosis = [(CFUs at 2 h)/(CFUs at 0 h)] X 100</li>
	<li>To measure the rate of intracellular replication of C. glabrata cells in THP-1 macrophages, collect intracellular yeast at different time points post infection i.e.&#160;4, 6, 8, 10, 12, and 24 hr, by repeating steps 3.3-4.3.</li>
	<li>Calculate fold survival/replication of C. glabrata cells in THP-1 macrophages by dividing total CFUs at any given time point with those at 2 hr (phagocytosed yeast).</li>
</ol>
5. Monitoring of Intracellular Replication Using Confocal Laser Scanning Microscopy
<ol>
	<li>Following the steps described in Section 1, seed PMA-treated 5 x 105 THP-1 cells in each well of two four-well chamber slides and incubate at 37 &#176;C with 5% CO2 for 12 hr.</li>
	<li>Replace old medium with prewarmed RPMI-1640 complete medium and allow cells to recover from PMA treatment for 12 hr.</li>
	<li>Prepare GFP (green fluorescent protein)-tagged C. glabrata cell suspension as described in Section 2 and infect to THP-1 macrophages to a MOI of 1. If C. glabrata strains expressing GFP are not available, yeast cells labeled with FITC (fluorescein isothiocyanate) can&#160; be used to monitor early events post infection viz.&#160;phagocytosis rate and phagolysosomal maturation at 2 hr.</li>
	<li>Incubate slides for 2 hr at 37 &#176;C with 5% CO2, invert them carefully to discard medium and wash 3x with sterile, prewarmed PBS.</li>
	<li>Add 500 &#181;l prewarmed RPMI-1640 complete medium to each chamber of one slide and incubate it at 37 &#176;C with 5% CO2 for 22 hr.</li>
	<li>To fix 2 hr C. glabrata-infected THP-1 macrophages, add 500 &#181;l of 3.7% formaldehyde (prepared in PBS) to each chamber of the other slide and incubate it at room temperature for 20 min in the dark.</li>
	<li>Wash slide thrice with PBS, add 500 &#181;l Triton-X (0.7%), and incubate at room temperature for 5 min in dark.</li>
	<li>Wash slide 3x with PBS, remove chamber from culture slide and air dry it for 3-5 min in the dark.</li>
	<li>Carefully mount a coverslip using Vectashield mounting medium containing DAPI (4&#39;,6-diamidino-2-phenylindole) on slide avoiding bubble formation. Remove gently the excess liquid with Kimwipe and seal coverslip edges with fingernail polish. Store slide in the dark at 4&#176;C until use.</li>
	<li>Repeat steps 5.4 and 5.6-5.9 to process THP-1 cells 24 hr post infection.</li>
	<li>Image cells using a laser scanning confocal microscope (60X oil-immersion objective, excitation at 405 nm and 488 nm for DAPI and GFP stain, respectively).</li>
</ol>

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The authors declare that they have no competing financial interests.

Innate immune system plays an important role in the control of opportunistic fungal infections. Macrophages contribute to antifungal defense by ingestion and destruction of the fungal pathogen. Thus, elucidation of factors that are required for survival and/or counteracting the antimicrobial functions of macrophages will advance our understanding of fungal virulence strategies. In this context, we have established an in vitro cell culture model system using macrophages derived from a human monocytic cell line TH...

Candida species are the leading cause of life-threatening invasive fungal infections in immunocompromised patients1. Candida glabrata, an emerging nosocomial pathogen, is the second or third most frequently isolated Candida species from Intensive Care Unit patients depending upon the geographical location1-3. Phylogenetically, C. glabrata, a haploid budding yeast, is more closely related to the non pathogenic model yeast Saccharomyces cerevisiae than to pathogenic Candida spp. including C. albicans4. Consistent with this, C. glabrata lacks some key fungal virulence traits including mating, secreted proteolytic activity and morphological plasticity4-5.
Although C. glabrata does not form hyphae, it can survive and replicate in murine and human macrophages6-8 suggesting that it has developed unique pathogenesis mechanisms. Limited information is available about the strategies that C. glabrata employs to survive nutrient-poor intracellular macrophage environment and counteract oxidative and nonoxidative host responses mounted by host immune cells5. A pertinent macrophage model system is a prerequisite to delineate the interaction of C. glabrata with host phagocytic cells via functional genomic and proteomic approaches. Peripheral blood mononuclear cells (PBMCs) and bone marrow-derived macrophages (BMDMs) of human and murine origin, respectively, have earlier been used to study the interaction of C. glabrata with host immune cells7,9. However, difficulty in obtaining PBMCs and BMDMs, their limited life span and intrinsic variation among different mammalian donors restrict the utilization of these cells as versatile model systems.
Here, we describe a method for establishment of an in vitro system to study the intracellular behavior of C. glabrata cells in macrophages derived from human monocytic cell line THP-1. The overall goal of this protocol was to enact a simple, inexpensive, quick, and reproducible cell culture model system that can be easily manipulated to study different aspects of host-fungal pathogen interaction.
THP-1 cells have previously been used to decipher the host immune response against a wide range of pathogens including bacteria, viruses, and fungi10-12. Monocytic THP-1 cells are easy to maintain and can be differentiated, upon phorbol ester treatment, to macrophages which mimic monocyte-derived macrophages of human and express appropriate macrophage markers13. The main advantages of THP-1 macrophage model system are the ease-of-use and the lack of sophisticated equipment requirement.
The protocol presented here is easily adaptable to study the interaction of other human fungal pathogens with host immune cells. The current procedure can also be employed to identify virulence factors for the pathogen of interest using high throughput mutant screens. This proof-of-concept was exemplified by the successful use of THP-1 culture model system to identify a set of 56 genes that are required for survival of C. glabrata in human macrophages8.

<table border="0" cellpadding="0" cellspacing="0" width="596">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="39">
			<td height="39" style="height:40px;width:163px;">THP-1</td>
			<td style="width:105px;">American Type Culture Collection</td>
			<td style="width:85px;">TIB 202</td>
			<td style="width:243px;">Human acute monocytic leukemia cell line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">RPMI-1640</td>
			<td style="width:105px;">Hyclone</td>
			<td style="width:85px;">SH30096.01</td>
			<td style="width:243px;">For maintaining THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Phorbol 12-myristate 13-acetate</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">P 8139</td>
			<td style="width:243px;">Caution: Hazardous</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">YPD&#160;</td>
			<td style="width:105px;">BD-Difco</td>
			<td style="width:85px;">242710</td>
			<td style="width:243px;">For growing Candida glabrata cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Formaldehyde</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">F8775</td>
			<td style="width:243px;">For fixation of C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Phosphate buffered saline (PBS)</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,&#160; pH 7.4) for washes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Saline-sodium citrate (SSC)&#160;</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (3 M NaCl, 0.3 M sodium citrate for 20x concentration) for washes</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Prehybridization buffer</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (50% formamide, 5x Denhardt&#8217;s solution, 5x SSC, 1% SDS) for hybridization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">VECTASHIELD mounting medium</td>
			<td style="width:105px;">Vector Labs</td>
			<td style="width:85px;">H-1200</td>
			<td style="width:243px;">For mounting slides for confocal microscopy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">32P-labeled &#945;-dCTP</td>
			<td style="width:105px;">JONAKI-BARC</td>
			<td style="width:85px;">LCP-102</td>
			<td style="width:243px;">For radiolabeling of signature tags</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">100 mm tissue culture dishes</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">430167</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">24-well tissue culture plate</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">3527</td>
			<td style="width:243px;">To perform C. glabrata infection studies in THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">4-chamber tissue culture-treated glass slide</td>
			<td style="width:105px;">BD Falcon</td>
			<td style="width:85px;">REF354104</td>
			<td style="width:243px;">To image C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hemocytometer</td>
			<td style="width:105px;">Rohem India</td>
			<td style="width:85px;"></td>
			<td style="width:243px;">For enumeration of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Table top microcentrifuge</td>
			<td style="width:105px;">Beckman Coulter</td>
			<td style="width:85px;">Microfuge 18</td>
			<td style="width:243px;">For spinning down cells in microtubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Table top centrifuge</td>
			<td style="width:105px;">Remi</td>
			<td style="width:85px;">R-8C</td>
			<td style="width:243px;">For spinning down cells in 15 ml tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Spectrophotometer</td>
			<td style="width:105px;">Amersham Biosciences</td>
			<td style="width:85px;">Ultraspec 10</td>
			<td style="width:243px;">To monitor absorbance of yeast cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Plate incubator</td>
			<td style="width:105px;">Labtech</td>
			<td style="width:85px;">Refrigerated</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Shaker incubator</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:85px;">Innova 43</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Water jacketed CO2&#160; incubator</td>
			<td style="width:105px;">Thermo Electron Corporation</td>
			<td style="width:85px;">Forma series 2</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Confocal microscope</td>
			<td style="width:105px;">Carl Ziess</td>
			<td style="width:85px;">Ziess LSM 510 meta&#160;</td>
			<td style="width:243px;">To observe C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Compound microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:85px;">CKX 41</td>
			<td style="width:243px;">To observe C. glabrata and THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">PCR machine</td>
			<td style="width:105px;">BioRad&#160;</td>
			<td style="width:85px;">DNA Engine</td>
			<td style="width:243px;">To amplify unique tags from input and output genomic DNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hybridization oven</td>
			<td style="width:105px;">Labnet</td>
			<td style="width:85px;">Problot 12S</td>
			<td style="width:243px;">For hybridization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">PhosphorImager</td>
			<td style="width:105px;">Fujifilm</td>
			<td style="width:85px;">FLA-9000</td>
			<td style="width:243px;">For scanning hybridized membranes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Thermomixer</td>
			<td style="width:105px;">Eppendorf</td>
			<td style="width:85px;">Thermomixer Comfort</td>
			<td style="width:243px;">For denaturation of radiolabeled signature tags</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:163px;">Gel documentation unit</td>
			<td style="width:105px;">Alphainnontech</td>
			<td style="width:85px;">Alphaimager</td>
			<td style="width:243px;">To visualize ethidium bromide-stained DNA in agarose gels</td>
		</tr>
	</tbody>
</table>

establishment of an in vitro system to study intracellular behavior of candida glabrata in human thp-1 macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Infection analyses of PMA-treated THP-1 macrophages with C. glabrata wild type (wt) cells revealed that wt cells were phagocytosed by macrophages at a rate of 55-65% after 2 hr coincubation. Further, C. glabrata cells were able to resist killing by THP-1 macrophages and underwent a moderate 5- to 7-fold increase in CFUs after 24 hr of coculturing with THP-1 macrophages8. &#160;Intracellular replication of wild type cells, transformed with GFP-expressing plasmid, in THP-1 mac...

Watch this Scientific Journal Video about Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages at JoVE.com

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

establishment-an-vitro-system-to-study-intracellular-behavior-candida

Research

JoVE Journal

Immunology and Infection

11.5K Views.  Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India. The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Video: Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

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

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

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.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...