This protocol is significant because it quantitatively measures phagocytosis of labeled Aspergillus fumigateurs Conidia by human leukocytes, along with the attachment of Conidia to cells, and lack of interaction by flow cytometry. The main advantage of this method is that it facilitates the fast analysis of a large number of cells. In addition, labeling of the cell markers allows to separate assessment of neutrophils and monocytes within the same sample.
In any this technique can be used to analyze other clinical relevant fungi like Mucorales. Instead red blood cells might be used as phagocytes. To begin this procedure, wet a paper towel with disinfectant, and place it into a bio safety cabinet.
Place a plate of grown Aspergillus fumigatus on top of the paper towel to prevent over-distribution of volatile Conidia. Add 10 milliliters of PBS containing 0.01%detergent on top of the fungus, and use a Drigalski spatula to spread the liquid over the plate, and rub off the dark colored Conidia. Transfer the Conidia suspension to a 50 milliliter through a 30 micrometer strainer, which will remove any residual mycelia.
Add another 10 milliliters of PBS with detergent to the plate, spread it with a spatula, and transfer it to the same tube through the strainer. Next, prepare a sterile solution of 0.1 molar sodium carbonate, dissolved in PBS. And prepare a 0.1 millimolar solution of FITC powder, in this sodium carbonate solution.
Re-suspend 100 million or less Conidia, in five milliliters of this FITC solution, in a 15 milliliters tube. Incubate in a rotator at 37 degrees Celsius for 20 minutes. To swell to Conidia, resuspend the FITC labeled Conidia in five milliliters of RPMI medium, supplemented with 10%FCS.
And incubate in a rotator at 37 degrees Celsius for the desired time. In a bio safety cabinet, place five milliliters of buffy coat into a 50 milliliters tube. Fill up the tube with EL buffer, and invert three times.
Incubate horizontally for five to eight minutes, until the milky appearance of the mixture turns clear. Then, centrifuge at 300 times G, and at room temperature for 10 minutes. Discard the supernatant and resuspend the pellet in one milliliters of EL buffer by pipetting.
Add another 24 milliliters of EL buffer, and invert the tube several times. Centrifuge at 300 times G and at room temperature for five minutes. Discard the supernatant and resuspend the cells in one milliliters of RPMI media, supplemented with 10%FCS.
To begin, transfer two million leukocytes and four million FITC-labeled Conidia in 1.5 milliliters of RPMI supplemented with 10%FCS, to a 12-well culture plate. Include a control of cells only with no Conidia, and a control of cells with unlabeled Conidia. Incubate the plate in a humidified carbon dioxide incubator, at 37 degrees Celsius for the desired amount of time.
When the incubation is complete, use a cell scraper to harvest the cells, and transfer them to a 15 milliliters tube. First, put 100 microliters of each sample and controls into one well of a 96-well V-bottom plate. Add 150 microliters of PBS containing two millimolar of EDTA for washing.
For color compensation, place one million cells without Conidia for each color, in other wells of the plate. Include a well of cells that will be left unstrained. Add 150 microliters of PBS containing two millimolar of EDTA to each well for washing.
Next, cover the plate with an adhesive foil, and centrifuge at 300 times G and at room temperature for five minutes. Then, remove the foil and discard the supernatant by quickly and forcefully inverting the plate only once over either the sink, or a disposable paper towel. Resuspend the cells in 100 microliters of antibody mix, and mix well by pipetting.
For color compensations, resuspend the respective cells in 100 microliters of PBS containing two millimolar of EDTA, and add a single antibody type to each well, at the same amount used in the antibody mix. Cover with an adhesive foil, and incubate at room temperature in the dark for 20 minutes. After this, remove the foil and add 150 microliters of PBS containing two millimolar of EDTA to each well for washing.
Cover the plate with adhesive foil again. Centrifuge at 300 times G and at room temperature for five minutes. Then remove the foil, and discard the supernatant by quickly and forcefully inverting the plate over either a sink or disposable paper towel.
Resuspend the cells in 200 microliters of PBS containing 2 millimolar EDTA, and transfer the cell suspension from each well, into a separate round bottom tube. Start the flow cytometer, and let it warm up. In the acquisition software, create a new experiment, and set up and label the new samples.
Set up the parameters and the detectors for the appropriate fluorophores, as outlined in the text protocol. In this software, display the appropriate dot plots as outlined in the text protocol. Using the cells only sample, acquire some cells and set the gate around leukocytes.
Based on the leukocyte gate, gate for CD45 positive cells, to separate from Conidia in the SSC CD45 plot. In a dot plot, CD14, CD66b, gate neutrophils and monocytes separately. After this, change from the cells only sample, to unlabeled Conidia.
In a dot plot, anti-FITC, APC FITC, display neutrophils in set quadrants for anti-FITC, and FITC signals. Open the statistics view and adjust the quadrants, allowing a maximum of 1%of cells in the quadrants Q1, Q2, and Q4.Repeat this process for the monocyte gate. In the leukocyte gate, record all samples with at least 20, 000 events.
Phagocytosis of FITC-labeled Conidia by human neutrophils can be read from the statistics views. In this study, a fast flow cytometric method is used to measure the interaction of Aspergillus fumigateurs Conidia with a large number of primary human leukocytes. Using the appropriate antibodies and the gating strategy shown here, a general gating of human leukocytes by FSC and SSC characteristics, is followed by a separation of leukocytes in free Conidia, by the pan leukocyte marker, CD45.
Conidia can reach almost cell size at the time of flow cytometry, especially when using swollen Conidia, and or long incubation times, and thus can buy us analysis. Since human primary monocytes and neutrophils take up Conidia differently, this protocol allows for the separate analysis of these cell population based, on staining with the well-established cell lineage markers, CD14 for monocytes, and CD66b for neutrophils. The percentage of human primary phagocytes internalizing Conidia can be highly variable among blood donors, but also depends on experimental factors, such as incubation time and swelling state of Conidia.
Spore internalization can be detected after 0.5 hours of co-incubation, and increases with time. Pre-swollen Conidia are taken up easier than resting spores, even at short incubation times. If Conidia are fixed with formaldehyde, phagocytosis is diminished compared to native Conidia.
Following this procedure, supernatants of co-incubated immune cells and Conidia can be collected and analyzed by Eliza to check if the interaction elicits a cytokine release by the immune cells. Please remember, human blood can potentially transmit infectious diseases. This work requires vaccination across Hepatitis B, the use of a bio safety cabinet, as well as wearing gloves at a lab coat.
