The overall goal of this method is to investigate changes in the lipid composition of neutrophils during the release of neutrophil extracellular traps by using thin layer chromatography and HPLC. This method can be used to gain more detailed information about the mechanisms that are involved in the formation of neutrophil extracellular traps, or shortly named, NET formation. The main advantage of this procedure is that it provides information on the role of lipids in the general function of neutrophils by utilizing an easy to follow protocol.
The isolation of human blood-derived cells will be demonstrated by a former PhD student of our lab, Ariane Neumann. And she will be assisted by Friederike Reuner. The preparation of cells for lipid analysis will be demonstrated by Graham Brogden a post-doc in my laboratory and Diab Husein, a master student in the biochemistry program.
To isolate human blood-derived neutrophils layer approximately 20 mL of blood onto 20 mL of sodium diatrizoate/dextran solution near a flame, without mixing. Centrifuge for 30 minutes at 470 times g without braking. It is critical not to mix the blood phase with the density grade in solution.
After the centrifugation step plus mononuclear cells need to be carefully removed to avoid contamination or unspecific activation of cells. Remove the mononuclear cells and the yellowish plasma layer. Transfer the polymorphonuclear cell, or PMN phase, into a new 50 mL tube, and fill it up to 50 mL with 1X phosphate buffered saline.
Centrifuge the sample for 10 minutes at 470 times g with braking. Then, remove the supernatant and re-suspend the pellet in 5 mL of sterile water for five seconds to lyse the erythrocytes. Immediately fill up the tube to 50 mL with 1X PBS and centrifuge the sample.
Upon removing the supernatant, the pellet should be white. If it is still red, repeat these steps. Then re-suspend the pellet in 1000 microliters of Roswell Park Memorial Institute, or RPMI medium.
Count the cell number using Trypan Blue staining and a hemocytometer under a light microscope. Prepare a cell suspension in RPMI at a concentration of two million cells per mL. Approximately 25 million neutrophils can be harvested from 20 mL of blood.
To pharmacologically treat neutrophils for lipid analysis use 15 million of the prepared cells. Incubate the samples in the reaction tubes for two hours at 37 degrees Celsius and 5%carbon dioxide. Following centrifugation of the samples remove the supernatant, and re-suspend the cell pellet in 300 microliters of HBSS.
After centrifuging the sample again using the same parameters, re-suspend the cell pellet in 1 mL of a 1:1 chloroform:methanol solution. Then homogenize the re-suspended pellet with a 26 gauge canula by pushing the sample in and out of a 1 mL syringe 10 times. To isolate the lipids from the human peripheral blood-derived neutrophils, take the prepared neutrophils and place them on ice.
Pipette the neutrophils to a 15 mL screw-cap glass tube with a PTFE seal, and homogenize them by shaking for one minute. Then add 2 mL of methanol followed one minute later by 1 mL of chloroform. After shaking for another one minute, rotate the glass tubes at room temperature and 50 rpm for 30 minutes.
Next, pellet the protein fraction by centrifuging the solution at 7 degrees celsius and 1, 952 times g for 10 minutes. Carefully decant the supernatant into a new 15 mL glass screw-cap tube, leaving the protein-containing pellet behind. Store the pellet at 20 degrees celsius for future quantification.
Add 1 mL of chloroform and wait one minute. Then, add 1 mL of double distilled water and invert the glass screw-cap tube with the sample for 30 seconds. After centrifuging the sample as before, remove and discard the upper phase down to, but not including the cloudy layer.
Dry the samples in a vacuum concentrator at 60 degrees celsius and store them at 20 degrees celsius until required. To begin, fill up to 5 mL of each solution in the corresponding glass chamber. Add any kind of filter paper to increase the running speed in each chamber.
Pre-incubate a 20x10cm HPTLC silica gel 60 glass plate in the first running solution. Once the running solution reaches the top of the plate dry it for 10 minutes at 110 degrees celsius. Dissolve the lipid pellet, previously obtained after drying with a vacuum concentrator, in the desired volume of 1:1 chloroform:methanol solution, and incubate for 15 minutes at 37 degrees celsius to dissolve.
Next use a ruler and a soft pencil to mark the loading spots for the desired number of samples plus at least one standard. Mark the running distance at approximately 4 cm for the first running solution and at approximately 6 cm for the second running solution. To load the samples, wash the 10 microliter syringe three times in 1:1 chloroform:methanol prior to loading each new sample.
Load 10 microliters of each sample drop-wise trying to concentrate the sample on as small an area as possible. Place the plate vertically into the first chamber with running solution one. Ensure that the plate is parallel to the wall of the glass chamber to achieve a uniform migration speed.
Take care that the plates are placed parallel to the back wall of the glass chambers and that the solvent fronts do not run too far to ensure that the lipids are efficiently separated. Once the solvent line has reached the first mark, remove the plate, dry it, and place it in the second solution. Repeat similar steps for the second and for the third running solutions.
Leave the plate in the solution until the solvent front reaches the top of the plate. Then, remove the plate, and dry it at room temperature for one minute. Place the plate in the copper sulfate solution for seven seconds.
After drying the plate thoroughly, bake it in an oven for seven minutes at 170 degrees celsius. Remove the plate from the oven and allow it to cool. To perform HPLC, attach the column to a guard cartridge on the instrument, and heat it to 32 degrees celsius.
Use methanol as the mobile phase at a flow rate of 1 mL per minute and 65 bar. Set the UV detector to measure at 202 nanometers to quantify the amount of cholesterol in each sample. Wash the HPLC machine thoroughly prior to analyzing the samples and establish a cholesterol standard curve as described in the text protocol.
Finally, re-suspend the samples in 500 microliters of 1:1 chloroform:methanol in amber colored 1.5 mL glass bottles with screw top red PTFE/white silicone lids before loading them on the HPLC instrument. Shown here is the lipid profile of neutrophils on an HPTLC plate. Lipids can be identified by comparing migration distances to the standard.
Also shown is an HPLC chromatogram showing two large peaks. The first peak just before two minutes is chloroform. The second large peak at five minutes is cholesterol.
Shown here are cholesterol standard curves determined by HPLC and by HPTLC. The standard curve obtained by HPLC is linear over a broader concentration range as compared to the HPTLC standard curve. After watching this video you should have a good understanding of how to isolate human blood-derived neutrophils and subsequently analyze their lipid composition.
While attempting this procedure it is important to remember to wear nitrile gloves to protect your hands against hazardous chloroform:methanol exposure and also to work under a fume hood. Future experiments will provide further information about the mechanisms involved in NET formation and general neutrophil functions which are still not fully understood. Don't forget that working with primary human blood-derived cells requires ethical permission as well as appropriate safety precautions such as working in a respective bio-safety lab to avoid risk exposure to human pathogens.
Finally the aim of our study is to find new therapeutic targets against an infection based on the innate immune system, and especially the lipid composition is very interesting for us since it will help us to understand why the neutrophil is acting antimicrobial against an infection.
