Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

The overall goal of this procedure is to elucidate a role for cholesterol in the uptake of cryptococcus neoformans by macrophages. This is accomplished by first depleting cholesterol from a murine macrophage cell line using methyl beta cyclodextrin. Next cholesterol depletion is confirmed and quantified by using thin layer chromatography and a commercially available kit.

Then the crypto caucus neoformans cells are ionized with antibodies specific to gluc, xlo manin or GXMA component of the cryptococcus capsule. Finally, the cholesterol depleted macrophages are infected with opsonize crypto occus neoformans. Ultimately, gem sustaining and light microscopy are used to visualize the infected cells and allow the degree of phagocytosis to be calculated.

The main advantages of this technique is that it can be done with basic lab equipment in a relatively short amount of time and still obtain quantitative results. This method can help answer key questions in the field of fungal pathogenesis. For example, what is the role of host lipid grafts in the optic of fungal pathogens?

The implication of this technique extend toward the therapy or the nuis of occus infection because the internalization within microphages is a critical step for the initiation of critical causes. Though these methods can provide insight into the infectivity of fungal pathogens, it can also be applied to other organisms, such as in obligate intracellular pathogens In a sterile biosafety cabinet seed 10 to the fifth, J 7 74 A 0.1 macrophage like cells per well of a 96 well culture plate. In 200 microliters of DMEM supplemented with 10%FBS and 1%penicillin streptomycin or PS incubate overnight at 37 degrees Celsius and 5%CO2 the following day.

Remove the medium from the cell monolayer and use filtered or autoclaved PBS to wash the cells twice. Add 200 microliters of methyl beta cyclodextrin or embeta CD solution at the desired concentration or one XPBS as a control and incubate for 30 minutes at 37 degrees Celsius with shaking. Remove the supernat and reserve it at room temperature for quantitative analysis with a commercially available kit.

Immediately following this procedure with PBS or serum free DMEM, wash the cells two to three times before carrying out cholesterol quantification or continuing to the infection. Step to infect the cholesterol depleted macrophages in a sterile biosafety cabinet. Seed 10 to the fifth macrophage like cells per well.

In a 96 well plate incubate overnight at 37 degrees Celsius and 5%CO2 at the same time. Grow a culture of C neoformans H 99 by isolating one colony from a struck plate and inoculating 10 milliliters of YNB incubate overnight at 30 degrees Celsius with shaking the following day. Centrifuge the C neo four mans culture at 1, 700 times G and four degrees Celsius for 10 minutes.

Remove the supernatant and add five milliliters of one XPBS before spinning again. Then use filtered one XPBS to wash the cells three more times before resus suspending the cells in five milliliters of one XPBS. Next, to prepare a serial dilution of the washed culture, add 100 microliters of the original sample to 900 microliters of one XPBS.

To obtain a one to 10 dilution, add 100 microliters of the one to 10 diluted sample to 900 microliters of one XPBS to obtain a one to 100 dilution. And finally, add 200 microliters of the one to 100 diluted sample to 800 microliters of one XPBS. To obtain a one to 500 dilution, add 10 microliters of the final dilution to a hemo cytometer and count to calculate the number of cells to prepare a working solution for activating macrophages and opsonizing c neoformans dilute LPS and interferon gamma 100 times from stock solutions by diluting 10 microliters of each compound in 990 microliters of PBS For each sample combines 7.5 microliters of LPS 1.25 microliters of interferon gamma 1.25 microliters of GXM antibody and 1.25 times 10 to the fifth.

See neo forman cells then using DMEM supplemented with FBS and Ps, bring the volume up to 250 microliters per sample. Vortex the solution and incubate for 20 minutes at 37 degrees Celsius with shaking prior to infecting the macrophages cholesterol depletion can be performed after cholesterol depletion. Use serum free DMEM to wash them twice before adding 200 microliters of Opsonize C neoformans working solution to each.

Well incubate for two hours at 37 degrees Celsius. Then to fix and stain the cells. After using DMEM to wash the cells twice and air drying the cell monolayer for 10 minutes, add 200 microliters of ice cold methanol fix for 15 minutes at room temperature before removing any remaining methanol.

Next, add 200 microliters of 10 x giza and incubate for five minutes at room temperature. Then use deionized water to wash two or three times, leave the cap off and dry overnight the next day or up to one week later under a microscope. Count 300 cells per data point and note the number of infected macrophages and number of engulfed cryptococcus cells.

Calculate the PHA acidic index by multiplying the percentage of an infected macrophages by the mean number of C neoformans per macrophage. Normalize the F acidic index by expressing values as a percentage of the one XPBS treated control. After several trials, calculate the mean value and the standard deviation of the mean to determine trends in F acidic index.

Use the student's T-test to determine significance as shown here. Analysis of the supernatant reserved from embeta CD treated cells revealed elevated cholesterol as compared to the supernatant isolated from PBS control cells, indicating a higher level of cholesterol depletion in the M beta CD treated cells. Depletion can be calculated using cholesterol values obtained from the supernatant and cell lysate as illustrated here to support cholesterol depletion results.

Cell lysate analyzed using TLC shows a marked decrease in staining of cholesterol in cells treated with increasing concentrations of M beta CD cytometry. Analysis of the TLC shows a similar trend to the quantitative assay following infection cells remain adhered and intact, and cell morphology remains unchanged in seen neoformans infected cells compared to untreated control cells. Micrographs of optimally infected cells clearly show c neoformans engulfed within the mammalian cells.

This graph demonstrates that when calculated from 300 macrophage cells per treatment group, a reduction in F acidic index was found in cholesterol depleted cells Once mastered, this technique can be performed in approximately five hours. This is not including incubation times that must be done overnight Following this procedure. Other methods such as extraction of detergent resistant membranes from the macrophages can be performed in order to answer additional questions, such as is cholesterol depletion reducing the optic of cryptococcus through affecting host lipid grafts After its development.

This techniques and pave ways to, for researchers in the field of membrane biology and immunology, to understand the mechanisms of phagocytosis by macrophage and pathogen interactions in pathogen fungi. Don't forget that working with pathogen can be extremely dangerous, and precautions such as wearing protecting equipment should always be taken while performing this procedure.