The overall goal of the following experiment is to study the effects of staphylococcal phenol, soluble modlin or PSMs on human neutrophils. This is achieved by first isolating neutrophils from human blood for subsequent in vitro analysis. Next, the neutrophils are either loaded with fluorescent dye and mixed with a concentration series of PSMs with and without inhibitors, or they're allowed to phagocytose S reus bacteria containing A PSM alpha promoter GFP reporter construct.
Ultimately activation of the neutrophils by PSM stimulation can be monitored by flow cytometry and expression of GFP by the phagocytosed. Bacteria can be analyzed by fluorescence microscopy By this method. You can study the effects of staphylococcal, phenyl, soy, molins, or neutrophils.
Of course, this method can be applied to many other cell types, stimuli and antagonist. We first had the idea for the microscopy part of this method when discussing a way to observe the effects of PSMs inside neutrophils. Using a bacterial GFP expression construct and time-lapse microscopy allows us to do exactly that.
After drawing five tubes of heparinized venous blood, pour 12 milliliters of a 1.119 grams per milliliter density fial solution into a 50 milliliter tube, and then carefully overlay 10 milliliters of a 1.077 grams per milliliter density FI call solution. Next, dilute the blood with an equal volume of PBS and layer 20 to 25 milliliters of blood carefully onto each dual layer fi call gradient. Centrifuge the gradients for 20 minutes at 396 times G and 22 degrees Celsius without breaking.
And then use a vacuum pump fitted with a sterile pipette tip to aspirate the top yellow plasma and PBMC layers. And then the third white layer. Now use a small plastic pipette to transfer the polymorphonuclear neutrophil or PMN fraction from each tube into a new 50 milliliter tube.
Add freshly prepared pre-cool our PMI supplemented with human serum albumin or R-P-M-I-H-S-A to each tube to bring the total volume to 50 milliliters. Then spin down the cells, aspirate the supernatant with a vacuum pump and gently vortex the red erythrocyte and PMN containing pellet. Next, add nine milliliters of sterile deionized water to the cells and start a timer.
Add exactly 30 seconds. Add one milliliter of 10 times concentrated PBS to stop the hyperosmotic shock. Then wash the cells in a total volume of 50 milliliters of cold R-P-M-I-H-S-A.
After removing the supernatant, add one milliliter of R-P-M-I-H-S-A to each palate, which is now white due to lysis of the erythrocytes. Then resus, suspend by pipetting and combine the pellets in one tube. Now count the cells and adjust the concentration to one times 10 to the seventh cells per milliliter.
Now load five times 10 to the sixth cells per milliliter with two micromolar flu oh 3:00 AM in R-P-M-I-H-S-A. And cover the tube with aluminum foil to protect it from light. Next, incubate the cells for 20 minutes at temperature under agitation.
In the meantime, prepare a threefold serial dilution of the stimulus at 10 times the final concentration. Then pre incubate 30 microliters of the stimulus with an equal volume of freshly prepared 10 times concentrated inhibitor or buffer for 10 minutes at room temperature. Next, wash the cells in 10 milliliters of R-P-M-I-H-S-A and resuspend the cells in fresh R-P-M-I-H-S-A at five times 10 to the sixth cells per milliliter.
Right before flow analysis, dilute the cells to two times 10 to the sixth cells per milliliter in R-P-M-I-H-S-A and add 200 microliters of cells per fax tube. Being careful not to activate the cells by shaking or pipetting load the first tube on the flow cytometer. Wait three seconds and then start the acquisition after a fixed time period, for example, eight seconds, take off the tube and quickly add 50 microliters of the lowest dilution of the stimulus to the sample.
Immediately reload the tube and continue the acquisition. When a dilution series is finished, wash the flow cytometer needle with fresh R-P-M-I-H-S-A before measuring a new series. After running the last sample, analyze the data with flow cytometry analysis software to analyze bacterial GFP expression.
After PMN phagocytosis grow an S ous strain containing the reporter construct of interest overnight in broth, remove any GFP expressed during the overnight culture by diluting the cells to OD 6 60 0 0.01. Grow the culture to OD 6 60 0 0.1. Dilute the cells one to 30, and then monitor the growth until the culture once again reaches an OD six 60 of 0.1.
Then collect the bacteria by centrifugation. Wash the cells once in DPBS and Resus. Suspend the culture.
Add one to 10 of the original volume to obtain an OD six 60 of 1.0 or roughly five times 10 to the eighth CFU per milliliter. Now mix the bacteria with the freshly isolated PNS in R-P-M-I-H-S-A at a 10 to one ratio. Then add pooled human serum to a one 10th total end volume of pooled human serum within the cell solution.
Stimulate phagocytosis by shaking the cell mixture for 10 minutes at 37 degrees Celsius. Then dilute the bacteria loaded PMNs to five times 10 to the fifth cells per milliliter, and transfer 250 microliters of the cells into each well of an eight well chambered cover slip. Finally, image the cells on an inverted microscope equipped with a 40 x objective and encased in a dark environment chamber and acquire images at multiple locations in the cover slip chambers every five to 10 minutes in both the bright field and the GFP channel to follow the bacterial GFP production in real time, incubating neutrophils with a concentration series of synthetic PSM alpha three results in a dose dependent rapid activation of the cells as measured by calcium flux.
Pre incubation of the PSM alpha three peptide with 0.01%0.1%or 1%Human serum significantly inhibited the ability of the stimulus to elicit a calcium response. Again in a dose dependent fashion at 1%serum. Hardly any activation is visible at these PSM alpha three concentrations.
In these images, two neutrophils containing phagocytose bacteria containing A PSM alpha GFP reporter construct are shown. The bacteria start to fluoresce about one hour after phagocytosis indicating expression of GFP from the PSM Alpha promoter. Bacteria outside the neutrophils do not fluoresce indicating that PSM alpha expression is rapidly switched on only when the bacteria are phagocytosed.
When attempting this procedure is important to remember to always use fresh neutrophils for optimal results. After watching this video, I hope you have a good understanding of how to measure calcium flux by flow cytometry and how to measure bacterial gene expression inside eukaryotic cells using fluorescence microscopy.
