We have developed a method of isolating rat neutrophils under subsequently extracting NETs. This method offers optimized accessibility for conducting NETs related experiments in both rats and rat-derived cell lines. In our study we conducted a comparison between neutrophils that were isolated from the peripheral blood and bone marrow of rats.
Our findings highlight the superiority of bone marrow derived neutrophils, showcasing a substantial numerical advantage in isolated neutrophil count alongside their proficient capability to synchronize. Our bone marrow based method for isolating rat neutrophils and NETs fills a research gap by optimizing isolation techniques for these cell types. Unlike previous methods that primarily used peripheral blood our protocol targets bone marrow, increasing neutrophil and large yields, and offering a more accurate rat model for studying neutrophil and NET biology.
To begin the bone marrow harvest, soak a euthanized rat in 75%ethanol to sterilize the fur and skin. Lay the rat on its back and sever the lower limbs at the hip. To protect the femur head.
Use dissection scissors to cut through the ligament at the hawk joint. Then fold the knee joint backward to expose the femur and tibia. With a pair of forceps and scissors.
Carefully remove any muscles, tendons, and other tissues connected to the bones. Poke the marrow through both bone ends with a five millimeter needle syringe to loosen the marrow matrix. Now use a fresh syringe to flush out the bone marrow with 10 to 15 milliliters of RPMI media.
Centrifuge the bone marrow cells at 300 G for 10 minutes at 20 degrees Celsius. Discard the supernatant and resuspend the cells in five to 10 milliliters of red blood cell lysis buffer. After incubating the suspension, add four times the volume of HBSS to the mixture.
Then centrifuge the cells at 600 G for five minutes at room temperature, Use the resulting pellet for further use. Isolate the neutrophils first with rat bone marrow neutrophil isolation kit. Layer two milliliters of 80 to 55%per call reagent in a 15 milliliter centrifuge tube to create a density gradient.
Then pipette the isolated neutrophils or the resuspension solution of cells isolated with rat bone marrow neutrophil isolation kit into the tube. Centrifuge the tube at 800 G for 40 minutes at 20 degrees Celsius with a sterile pipette. Collect the 70%gradient layer, including the cell layers at the 65 to 70%boundary.
Transfer the cell suspension to a new 15 milliliter centrifuge tube. Add 15 milliliters of HBSS into the tube and gently invert to mix its contents. Finally, centrifuge the tube at 300 G for 10 minutes at room temperature to pellet the cells for the one step process.
After pipetting the bone marrow suspension directly into the density gradient tube centrifuge as demonstrated previously. Next, pipette out the 70%gradient layer, including the layers at the 75 to 70%gradient boundary. Transfer this cell suspension to a new 50 milliliter centrifuge tube with 10 milliliters of HBSS.
Then centrifuge the tube at 400 G for five minutes at room temperature to pellet the cells. Count the isolated neutrophils using a hemocytometer and assess the cell viability via trypan blue staining. The two-step method had an enhanced purity level of 90%as relative to the 50%achieved by the one step method.
To begin resuspend the isolated rat neutrophils in four milliliters of supplemented DAR PMI media in a sterile 10 centimeter Petri dish. Next, add four microliters of PMA to the neutrophil suspension to trigger the formation of NETs. Incubate the mixture at 37 degrees Celsius for three hours under 5%carbon dioxide.
For the negative control. Add four to five microliters of Dnase 1 to the neutrophil suspension to degrade the secreted NETs. Then add four milliliters of HBSS to wash the NETs.
Flush the plate with four milliliters of fresh culture media for each plate to detach the NETs. Now collect the washing medium in a new tube and pipette frequently to ensure complete resuspension of the NETs. Centrifuge the suspension at 300 G for 10 minutes at 20 degrees Celsius to remove any floating cells.
Then collect the supernatant medium with NETs in a new tube centrifuge, the suspension of NETs and floating cells. In a 24 well plate with 1.4 centimeter glass cover slips placed in it to settle any floating cells. Then fix the cells on cover slips with 4%PFA and proceed to verify the presence of the NETs.
Next place a drop of HBSS on a paraffin film covered test tube stand. Invert the cover slip into the HBSS droplets to wash it. After the wash, incubate the cells in 250 microliters of 0.5 x triton X 100 for 10 minutes at room temperature to permeabilize them.
Then wash the cells three times in HBSS for one minute. Seal the cells in 10%normal donkey serum at room temperature for one hour. Finally, incubate the cells with 70 to 90 microliters of anti rat antibodies overnight at four degrees Celsius.
Wash the cover slip in HBS S3 times for five minutes each. Now incubate the cover slip in the secondary antibodies at room temperature for one hour in the dark before washing an HBSS. Stain the cell nuclei and NET skeletons in 70 to 90 microliters of dappy.
Perform HBSS wash three times for five minutes each. To begin pipette 50 microliters of the isolated rat NET samples to a tube with 450 microliters of tris EDTA buffer. Now pipette 100 microliters of the DNA standards and the samples into a 96 well plate.
Add an equal volume of assay reagent into the wells. Then incubate the plate at room temperature for two to five minutes, avoiding exposure to direct light. Place the plate in a fluorescent microplate reader set at an emission spectrum of around 530 nanometers and an absorbent spectrum of about 480 nanometers.
Incubate the cells in one microliter of a nucleus stain for 30 minutes. Next, incubate the cells in one microliter of cell-free DNA strain for 10 minutes. Gently mix the fluorescent dies.
Load the sample plate into the cytometer, switch to cytox green channel. Then select the hexed channel. Set the illumination to blue 377/447, and adjust the exposure time to 300, 000 microseconds.
Click on focused setup and then click Register Auto. To automatically adjust the focus, select the OX screen channel and set the illumination to green 483/536. Set the exposure time for cytox screen to 30, 000 microseconds.
Click on start scan to begin the scanning process. Comparable responses in NET secretion were discernible between peripheral and bone marrow neutrophils. Irrespective of PMA stimulation, rat ness also exhibited limited cross-linking capabilities with one another.
Incubation with PMA led to a 10%increase in cell-free DNA content after four hours indicating a higher propensity for triggering NET formation.
