1. Cholesterol Depletion of J774A.1 Cells with MβCD
2. Observation of Cholesterol Content by Thin Layer Chromatography (TLC)
3. Infection of Macrophages with C. neoformans (H99)
4. Trypan Blue Assay
Cholesterol Depletion
Analysis of the supernatant reserved in step 1.3 of the protocol by following the manufacturer’s instructions in the Amplex Red Cholesterol Assay kit yields an elevated concentration of cholesterol in MβCD treated sample as compared to the 1x PBS control. Depending on cell type and MβCD concentration used cholesterol depletion may vary. For J774 treated with 10 mM MβCD, a depletion of approximately 50% was observed. Depletion can be calculated using values obtained from the supernatant and cell lysate collected in step 1.4 (Figure 1).
Cell lysate analyzed using TLC shows a marked decrease in staining of cholesterol in cells treated with increasing concentration of MβCD (Figure 2A). Densitometry analysis of the TLC shows a similar trend to the quantitative assay (Figure 2B). The Bligh-Dyer method gives a crude extract of total lipids and it is essential to allow for adequate separation of lipids in order to identify the correct band utilizing the cholesterol standard.
Infection
After following the infection procedure, cells remain adhered and intact. Cell morphology remains unchanged between treatment groups. A control group that has not been exposed to C. neoformans serves as a checkpoint (Figure 3). It is possible to obtain suboptimal results and may manifest as lysis of cells and other abnormal morphologies. The most likely cause is contamination of the cell line or reagents used in the procedure. Micrographs of optimally infected cells clearly show C. neoformans engulfed within the mammalian cells. Differences in number of phagocytized yeast may be noted by observation between treatment groups (Figure 4). After calculating phagocytic index from 300 macrophage cells per treatment group a reduction in phagocytic index is found in cholesterol depleted cells (Figure 5). The reduction in the phagocytic index does not appear to be dependent on potential differences in macrophage activation, although they may occur. Performing the infection in the absence of macrophage activators, but after treatment with MCD results in a similar reduction of phagocytic index (data not shown).
Trypan Blue
Trypan Blue staining is used to assess the viability of cells after cholesterol depletion. No change in viability is observed between PBS treated and 10mM MCD treated cells. Viability appears to drop off slightly after treatment with 30 mM MCD, which may be expected due to the approximately 75% depletion in cholesterol (an essential lipid) observed in the densitometry analysis (Figure 6 and Figure 2B).
Figure 1. Cholesterol content of supernatant after treatment. Quantification of cholesterol in the supernatant collected from treated cells shows enrichment in MCD when compared to 1x PBS. Cholesterol depletion is 50 ± 5% calculated from total cholesterol in 1x PBS (supernatant + cell lysate). Error bars show standard deviation (n = 5).
Figure 2. TLC of cholesterol in cell lysate and densitometry. Image of developed TLC plate visualized with MnCl2 charring. A marked decrease in cholesterol is seen after MβCD treatment (A). Densitometry analysis of bands as compared to the PBS treated control (shown as 100%) confirms trend found in Cholesterol quantification assay (B). Please click here to view a larger version of this figure.
Figure 3. Uninfected control micrographs of treated J774 macrophages. Images of uninfected J774 cells taken at 200X magnification. Scale bar is 50 μm. 1x PBS (A), 10 mM MβCD (B), and 30 mM MβCD (C) treated cells show no change. Please click here to view a larger version of this figure.
Figure 4. Infection of J774 macrophages with C. neoformans. Images of infected J774 cells taken at 400X (top row A.1 – C.1) and 1,000X (bottom row A.2 – C.2) magnification are shown. Internalized C. neoformans cells appear as blue-violet spheres with a lighter ring surrounding them. Cells treated with 1x PBS (A), 10 mM MβCD (B), and 30 mM MβCD (C) show differences in C. neoformans uptake. Please click here to view a larger version of this figure.
Figure 5. Phagocytic index. Phagocytic index is shown with respect to the control group that was treated with PBS (Marked at 100 for comparison). Phagocytic index was reduced by 25% by 10 mM MβCD treatment and by almost 55% by 30 mM treatment. Error bars show standard deviation of the mean (n = 4).
Figure 6. Cell viability. Variations in cell viability by trypan blue assay show little variation when comparing all three treatment groups. There is a slight drop off in viability in the 30 mM MβCD treatment group, which can be expected from depletion of such a major component of the membrane. Error bars show standard deviation (n = 4).
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Class II type A2 Biosafety Cabinet | Labconco | 3460009 | |
J774A.1 cell line | ATCC | TIB-67 | Arrives Frozen. See ATCC instructions for culturing. |
Dulbecco’s Modified Eagle Medium | Gibco | 11995-065 | Store at 4 °C and warm to 37 °C prior to use |
HI Fetal Bovine Serum Performance Plus | Gibco | 10082-147 | Keep frozen at -20 °C and thaw before adding to DMEM |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | Used to suplement DMEM |
Isotemp Cell culture incubator | Fisher Scientific | Model # 3530 | |
96-Well culture dish | Corning Inc. Costar | 3595 | |
10x Phosphate Buffered Saline | Fisher Scientific BioReagents | BP3994 | Dilute to 1x and filter or autoclave prior to use. |
Methyl-β-Cyclodextrin | Sigma Life Science | C4555-10G | Dissolve in 1x PBS to make solutions of 10mM and 30mM concentrations |
Orbital Shaker | Labline | ||
Amplex Red Cholesterol Assay Kit | Life Technologies Molecular Probes | A12216 | All reagents for Cholesterol Assay are contained within the kit. Follow Manufacturer instructions. |
96-Well Black Assay plate | Corning Inc. Costar | 3603 | |
FilterMax microplate reader | Molecular Devices | Model F5 | |
TLC Chamber | Sigma-Aldrich | Z126195-1EA | |
Chloroform | Sigma-Aldrich | 650498-4L | |
Methanol | Sigma-Aldrich | 34860-2L-R | |
TLC Paper | Whatman | 3030917 | Cut down to size needed for TLC tank |
Fume Hood | Any fume hood that complies with AIHA/ANSI Standards | ||
6-Well Plate | Corning Inc. Costar | 3506 | |
Trypsin-EDTA | Gibco | 25300-054 | |
Cell Scraper | Corning Inc. Costar | 3010 | |
Hemocytometer | Hausser Scientific | 1490 | |
Centrifuge | Beckman Coulter | Model Alegra x-30R | |
Votex Mixer | Fisher Scientific | 12-812 | |
Balance | Mettler Toledo | Model # MS104S Meaures down to .1 mg | |
Glass Pasteur Pipette | Fisherbrand | 13-678-20A | |
Cholesterol | Avanti Polar Lipids | 700000 | |
SpeedVac Concentrator | Thermo Scientific | Model # SPD2010 | |
Petroleum Ether | Fisher Scientific | E139-1 | |
Diethyl Ether | Sigma-Aldrich | 309966 | |
Acetic Acid | Sigma-Aldrich | 320099 | |
TLC Silica Gel 60 with concentrating zone | Analytical Chromatograhy Millipore | 1.11845.0001 | |
Iodine Chips | Sigma-Aldrich | 376558-50G | |
Sulfuric Acid | Sigma-Aldrich | 320501 | |
Manganese Chloride | Sigma-Aldrich | 244589 | |
UVP EC3 Imaging System | Ultra-Violet Products Ltd. | Use the Vision Works LS software for densitometry analysis | |
Glass Bottom Confocal Dish | MatTek | P35G-1.5-10C | www.glassbottomdishes.com |
Cryptococcus neoformans (H99) | Obtained from Duke University Medical Center | ||
YNB | BD | 239210 | See manufacturer for preparation instructions. Use a Glucose concentration of 20 g/L. |
Lipopolysaccharide | Sigma | L4391-1MG | Dissolve in 1x PBS to make 1mg/mL stock. Store at -20 °C. |
Interferon gamma | Sigma | I4777 | Dissolve in 1x PBS to make .1 mg/mL stock solution |
Glucuronoxylomannan antibody (anti-GXM) | Gift from Arturo Casadevall's Lab concentration is 1.98 mg/mL | ||
Giemsa | MP Biomedicals | 194591 | Dissolve .8 g of Giemsa in 25 mL of Glycerol and heat to 60 °C for 1 hour. Add 25 mL of methanol to the solution and allow to age at room temperature for at least 1 month. |
Microscope | Zeiss | Observer.D1 microscope with AxioCam MRm for taking images |
Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl–cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFNγ) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.
Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl–cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFNγ) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.
Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl–cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFNγ) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.