The overall goals of this method are to generate and identify in vitro cultured giant phagocytes derived from circulating human neutrophils for the investigation of their role in inflammatory and antiinflammatory immune responses. This method can be used to obtain and identify a new subpopulation of giant phagocytes which develop from human neutrophils to maintain long-term culture conditions. This technique can be used to further investigate the significance and functions of giant phagocytes as well as to answer key questions about their activation and plasticity.
Demonstrating the procedure will be Eva Leder, our research assistant, and Oksana Rogovoy, post doc, both from my laboratory. Use a sterile scalp vein set to obtain at least 40 milliliters of venous blood from a healthy young volunteer. Then gently mix the blood in a biosafety laminal flow hood.
Dilute 10 to 12 milliliter aliquots of the whole blood sample to a final volume of 24 milliliters with pre-warmed ION-free PBS containing 2%heat inactivated FCS. Then add 12%milliliters of polysucrose 1119 to the bottom of one 50 milliliter sterile polypropylene conical centrifuge tube per blood sample aliquot, followed by the careful layering of 12 milliliters of polysucrose 1077 on top of the polysucrose 1119 gradient solution. Carefully pipette 24 milliliters of the diluted blood onto individual tubes of the density gradient solutions and separate the cells by centrifugation.
Two resulting opaque layers should be observed. Discard the fluid up to 0.5 centimeters above the mononuclear cell layer in each tube. Then transfer the mononuclear cells from each sample to a single new tube.
Next, discard the remaining fluid up to 0.5 centimeters above the polymorphonuclear cell or PMN layer and transfer the PMNs from each sample into a different new tube. Wash the PMNs in a final volume of 30 milliliters of PBS containing 2%heat inactivated FCS followed by lysis of the red blood cells with three milliliters of sterile ice cold hypotonic 0.2%sodium chloride. After 30 seconds on ice, restore the isotonicity with three milliliters of sterile ice cold 1.6%sodium chloride.
Then add six milliliters of 37 degree Celsius RPMI 1640 Medium supplemented with heat inactivated FCS and collect the cells by centrifugation. Resuspend the pellet in four milliliters of RPMI 1640 supplemented with 10%heat inactivated FCS. After counting, adjust the concentration to 1.25 to 1.5 times 10 to the sixth PMN per milliliter of culture medium and plate one milliliter of cells per well into individual wells of a 24-well plate.
Then place the plate in a humidified 5%carbon dioxide incubator at 37 degrees Celsius, feeding the cultures by gentle replacement of half of the medium with fresh RPMI 1640 Medium supplemented with 10%heat inactivated FCS every three days. The neutrophils will differentiate into giant phagocytes within seven days of culture. On day seven of culture, carefully remove half of the medium from each well.
Then robustly pipette the remaining medium to remove the lightly adhered giant phagocytes. Transfer the detached cells from two to four wells of each treatment group into individual 15 millimeter conical tubes and collect the cells by centrifugation, resuspending the pellets in 100 to 120 microliters of medium per tube. Then equip two to three holders per treatment group with microscope slides, filter cards and funnels.
Next, add the entire volume of cells from each tube into the appropriate Cytospin funnel. Then load the holders onto a Cytospin and spin the cells onto the slides. Dry the spun slides for 10 minutes.
Then use a water stable marker to draw a hydrophobic barrier around the cells and fix the samples with 4%paraformaldehyde under a chemical hood at room temperature. After 10 minutes, briefly rinse the slides three times with about 100 microliters of PBS per wash. Then permeabilize cells with 0.5%Triton X-100 in PBS for 10 minutes at room temperature followed by five PBS washes as just demonstrated.
Block the nonspecific binding with 10%normal goat serum in RPMI 1640 Medium for the appropriate time period followed by a single PBS wash. Now, add a small amount of water to the box to maintain humidity and then label the cells with about 100 microliters of the appropriate primary antibody of interest overnight at four degrees Celsius in a humidified dark chamber. Next, perform a single PBS wash and then add the appropriate fluorescence conjugated secondary antibody.
After 40 minutes at room temperature protected from light, wash the samples to remove the excess antibody and mount the slides with one drop of mounting medium per sample and a coverslip. Analyze the slides by confocal laser scanning fluorescence microscopy within 30 to 120 minutes under a 40X emersion oil objective. Then calculate the cell area, fluorescence intensity and co-localization using the appropriate software.
Neutrophil development into giant phagocytes within seven days of culture can be observed in these images. Auto phagocytosis is evident as early as 90 minutes after neutrophil culture with fluorescent membrane stains with a vastly enlarged cell diameter apparent by days four to seven. If the neutrophil cultures are supplemented with GM-CSF and IL-4 however, the cells demonstrate an overall smaller diameter and cytoplasmic projections resembling dendritic cell-like cells.
Time lapse microscopy of the giant phagocytes on culture days three to four and days four to five reveals none or lightly adherent cells with a limited movement capacity that actively ingests the surrounding neutrophil remnants and debris. In this mixed monocyte neutrophil culture, the migration of an actively crawling macrophage can be compared to the nearly stationary movement of a fluorescently labeled giant phagocyte in the same culture well. The neutrophilic origin of giant phagocytes can be verified by their positive expression of no neutrophil markers and their lack of expression of monocytic and dendritic cell markers.
Giant phagocytes also generate basal reactive oxygen species and respond to zymosan and PMA stimulation by oxidative burst. However, unlike monocytes or neutrophils, they also respond by oxidative bursts to oxidized low density lipoprotein stimulation. Once mastered, this technique can be completed in six to seven days.
While attempting this procedure, it is important to remember to replace half of the medium gently when feeding the neutrophil cultures. Following this procedure, other staining techniques can be performed to answer questions about the additional functions of these intriguing cells in vitro as well as in vivo. This technique could pave the way for researchers in the field of neutrophil biology to explore neutrophil activation and plasticity and to identify these cells in vivo in physiological and pathophysiological conditions in humans.
After watching this video, you should have a good understanding of how to obtain and identify giant phagocytes in culture. Please don't forget that working with human blood can be potentially infectious and that precautions such as wearing gloves and discarding all fluids and use disposable equipment into the appropriate biohazard containers is a must when performing this procedure.
