The NeutroFun screen protocol provides a group of functional tests that use low-cost chemicals and can be performed in a single experiment from fresh neutrophils. This allows a preliminary evaluation of some major neutrophil functions to guide more laborious and expensive follow-up studies. Neutrophil research employs various modulation conditions, such as cytokines, peptides, microbiome products, and physical stimuli.
Additionally, analysis matters include flow cytometry, proteomics, micropick devices, and immunocytochemistry. The extensive brand operations and assays used in combination makes the work neutrophils research long, laborious, and expensive. Typically, this reduces the number of conditions tested or the number of tests performed.
The current state-of-the-art methods require expensive consumables, many cells, and lengthy preparation procedures. Our protocol, NeutroFun screen, offers a quicker and more cost-effective approach to evaluate cell functionality compared to other methods. This is achieved by combining various analysis within the protocol while maintaining accuracy and comprehensiveness in the results.
Two researchers perform this workflow simultaneously for a quick overview of the neutrophil function. Recenter one isolates the PNNs, adjusts concentration, and incubates the activation systems, whereas researcher two prepares materials for the subsequent tests, then researcher one performs phagocytosis, NET suggestive, and colorimetric NBT assays, while researcher two conducts realtime migration and NBT slide tests. Prepare 12 milliliter dilutions of 60%and 70%separation media in 50 milliliter conical tubes.
Next, prepare the gradient from bottom to top by adding four milliliters at a time of the 60%dilution over the 70%dilution using a five-milliliter pipette. Then, carefully layer 12 milliliters of heparinized blood on top of the density gradient and centrifuge at 200 G for 15 minutes at room temperature. Discard the plasma and mononuclear cell layers, then carefully transfer approximately 7.5 milliliters of the layer above the erythrocyte pellet into two 15 milliliter conical tubes.
Centrifuge the tubes at 300 G for five minutes at 19 degrees Celsius. Proceed to wash the cell pellet with Hanks'Balanced Salt Solution, or HBSS. Then, perform hypotonic lysis and remove the remaining red blood cells.
After discarding the supernatant, gently resuspend the polymorphonuclear neutrophils in the remaining buffer and transfer them to an ice-cold microcentrifuge tube. Next, transfer three one microliter aliquots of the cell suspension to a clean glass slide. Stain with fast panoptic to evaluate the cell morphology and purity.
Observe the cells under a microscope and count 300 random cells in each well. Mark the neutrophils and other cell types to assess the purity of the separation. Next, transfer one microliter of the cell suspension into 49 microliters of 0.2%trypan blue dye and count dead and viable cells using a Neubauer chamber.
Adjust the cell concentration to 6, 667 cells per microliter, with 50%autologous plasma and 50%HBSS, supplemented with calcium and magnesium. Then, divide the cell suspension evenly among 1.5 milliliter microcentrifuge tubes corresponding to the testing conditions, including the negative control. Prepare an activation system for every condition, ensuring the final cell concentration remains at 6, 600 polymorphonuclear neutrophils per microliter.
Incubate at 37 degrees Celsius without rotation. Begin by dissolving 0.0005 grams of NBT in 10 microliters of DMSO. After vortexing for 15 minutes, add 90 microliters of Hank's Balanced Salt Solution, or HBSS, with calcium and magnesium and one microliter of the test substances.
Again, vortex the tube for up to two minutes. To perform the NBT slide test, gently mix and transfer two microliters of the previously prepared activated polymorphonuclear neutrophils onto a clean glass slide. Incubate the slide in humidified chamber at 37 degrees Celsius for 20 minutes.
Next, add one microliter of the NBT working solution over the cells. Incubate again for 20 minutes, protecting from light. After drying the slide, fix it by applying a drop of methanol in each well for one minute.
Stain the slide with 0.03 safranin for an additional one minute. In each well, count 100 random cells, differentiating neutrophils with and without formazan deposits. To perform the NBT spectrometry assay, gently mix and transfer 90 microliters of the previously prepared activated polymorphonuclear neutrophils to a clean microcentrifuge tube, then carefully add 20 microliters of a six millimolar NBT solution and incubate the tube in the dark for 37 degrees Celsius for 20 minutes.
After incubation, add 100 microliters of 10%SDS and vortex. Sonicate with a tip sonicator at 60%amplitude, adding five cycles of 15 seconds each and 15-second intervals. Centrifuge at 12, 000 G for five minutes.
Transfer 60 microliters of supernatant to a clear bottom 96-well plate and measure the absorbance of the formazan product at 570 nanometers. The results of the spectrophotometric assay indicated that 100 nanomolar PMA induced elevated ROS production compared to the fMLP and control groups. The NBT slide test results corroborated the spectrophotometric results, showing PMA as the most intense ROS production-inducing stimulus compared to fMLP.
Add approximately 0.75 milligrams of dry yeast to 200 microliters of Hank's Balanced Salt Solution, or HBSS, containing calcium and magnesium ions. Next, incubate the mixture in a thermomixer at 100 degrees Celsius and 500 RPM for a minimum of 15 minutes. Following incubation, transfer five microliters of yeast suspension to 45 microliters of 0.2%trypan blue dye.
Count the yeast cells using a Neubauer chamber. Adjust the concentration of the initial suspension to 33, 000 yeast cells per microliter using HBSS with calcium and magnesium. Next, gently mix and transfer five microliters of the previously prepared activated polymorphonuclear neutrophils to five microliters of the yeast cell suspension in a new sterile microcentrifuge tube.
Immediately transfer six microliters of the polymorphonuclear neutrophils and yeast suspension into three wells of a clean glass slide, two microliters each, and incubate the slide in a humidified chamber for 40 minutes. After staining with fast panoptic, observe the cells under the microscope. Although both 100 nanomolar fMLP and 16 micromolar antimicrobial peptide induced an increased yeast engulfment, the difference was not statistically significant until the dual stimulation, which significantly enhanced the phagocytosis compared to the control group.
Prepare the chemotactic gradient by adding 160 microliters of chemoattractant to the lower chamber of an impedance-based realtime cell analyzer, or RTCA plate. For negative controls and blanks, add 160 microliters of Hank's Balanced Salt Solution, or HBSS, with calcium and magnesium. Attach the upper chamber and add 25 microliters of HBSS with calcium and magnesium.
Incubate at room temperature for at least one hour to form the chemotactic gradient. Next, gently mix and transfer 60 microliters of the previously prepared activated polymorphonuclear neutrophils into the upper chamber. Then, add 60 microliters of HBSS with calcium and magnesium to the blank.
Place the RTCA plate in the analyzer and program its software to measure the cell index every 60 seconds for two hours. Fit the curve with the obtained data. Real-time cell migration exhibited a chemotactic effect of 100 nanomolar fMLP on neutrophils.
Gently mix and transfer four microliters of the previously prepared activated polymorphonuclear neutrophil system under evaluation into two wells of a clean glass slide. Incubate the slide in a humidified chamber at 37 degrees Celsius for 30 minutes. Add one microliter of DNase I to one of the wells and incubate for 20 minutes at 37 degrees Celsius.
After staining with fast panoptic, evaluate under a microscope. Qualitative analysis of the panoptic-stained neutrophils showed that the control and fMLP groups did not show any indication of net release, while activation with PMA induced the formation of net-like structures, was degraded by DNase I treatment. The effects of antimicrobial peptide on the neutrophils showed that such a stimulus might be able to elicit net release, since the slide's presented net-like structures were degraded by DNAse I.
