This experiment uses flow cytometry to detect and quantify nuclear proteins in isolated and immuno labeled nuclei from neutrophils to purify the neutrophils from human peripheral blood. Remove the red blood cells with dextrin followed by density gradient centrifugation of the plasma. Then using anti receptor antibodies, stimulate the purified neutrophils via integrins or FC receptors.
Proceed to isolate the nuclei and fix the samples for immuno labeling with specific antibodies against the NU nuclear factor of interest. For example, NF kappa B finally analyze the nuclei by flow cytometry in order to detect small changes in nuclear factor activation. Results are obtained that show stimulation of neutrophils via their integrins leads to an increase in nuclear concentration of the transcription factor N of kappa B Signaling pathways that lead to nuclear factor activation are usually studied by reporter gene assays or by electrophoretic mobility shift assays in primary cells.
Often these methods are not suitable. Flow cytometry offers rapid detection and quantification of nuclear proteins in isolated nuclei. Start with 10 milliliters of freshly collected blood from healthy adult volunteers.
Pipette two milliliters of 6%dextrin T 500 solution into a 15 milliliter conical centrifuge tube. Then add 10 milliliters of blood mixed by inverting the tube two or three times and allow 45 minutes for erythrocyte sedimentation by gravity in a fresh 15 milliliter conical centrifuge tube. Place five milliliters of FI fial pack.
Harvest the leukocyte rich plasma that formed above sedimented erythrocytes. Carefully layer it on top of the fial pack to form two phases, centrifuge at 516 Gs for 20 minutes at four degrees Celsius. Remove the supernatant and break the cell pellet by tapping the tube against rack.
Then resuspend the cells in 10 milliliters of cold PBS. Transfer the cell suspension to a 50 milliliter conical centrifuge tube and centrifuge at 290 Gs for five minutes at four degrees Celsius. Break the cell pellet as before and add 10 milliliters of cold hypotonic solution to lye erythrocytes.
Mix by swirling the tube gently for exactly one minute. Next, add 10 milliliters of cold hypertonic solution mix and store on ice. Now enumerate the cells after pelleting the neutrophils by centrifugation.
We suspend the PMN at 10 to the seven cells per milliliter. In cold PBS keep the cells on ice. Aliquot 100 microliters of PMN suspension into 1.5 milliliter einor tubes.
Then add the corresponding monoclonal antibody at 10 micrograms per milliliter and incubate on ice for 15 minutes. To wash the cells, add one milliliter of cold PBS centrifuge and aspirate the supernatant. Then gently break the cell pellet and wash twice more with PBS.
To remove unbound antibody, re suspend the washed PMN in the same initial volume of warm PBS containing 60 micrograms per milliliter of go anti muse IgG incubate at 37 degrees Celsius for one to 20 minutes depending on the nuclear factor of interest. Now add one milliliter of cold PBS After pelleting the cells by centrifugation, remove the snat and freeze the cell pellets immediately in a dry ice ethanol bath. For each sample, remove the tube from the dry ice set ethanol bath wipe clean and resus.
Suspend the frozen PMN pellets in 100 microliters of cold hypotonic solution. Place all samples on ice to monitor samples for nuclei integrity. Stain an Eloqua of the nuclei suspension with trian blue.
If nuclei suspension contains many intact cells or debris, it is better to discard the preparation and start the procedure with another sample. After pelleting the nuclei by centrifugation, remove the supernatant very carefully then to fix the nuclei at 100 microliters of cold, 4%Paraform aldehyde solution and incubate the nuclei on ice. After pelleting the nuclei carefully remove the snat at 100 microliters of cold permeation buffer and incubate the samples on ice for 10 minutes.
Then harvest the perme nuclei by centrifugation. At this point, the nuclei pellets do not attach well at the bottom of the tube. It is recommended to centrif future again.
If the pellet gets loose To block non-specific binding sites, we suspend the nuclei in 500 microliters of cold, 4%fetal bovine serum in PBS and incubate on ice for 20 minutes. Pellet the nuclei by centrifugation, then resuspend the nuclei and 100 microliters of cold PBS containing 4%FBS and 2.5 micrograms per milliliter of the monoclonal antibody against the nuclear factor of interest. Incubate the samples on ice for 20 minutes after washing the samples twice with 500 microliters of cold PBS with 4%FBS, we suspend the nuclei in 100 microliters of cold PBS containing 4%FBS and 10 micrograms milliliter of the corresponding FZ labeled secondary antibody incubate on ice for 20 minutes after two washes, re suspend the nuclei in 400 microliters of cold 4%Paraldehyde in PBS.
Analyze the immuno labeled nuclei in a flow cytometer such as a fact scan or similar apparatus. Adjust the acquisition settings two FSC at log scale at 10 to the negative one SSC at log scale at 196 and gate nuclei in adopt plot. Acquire 10, 000 nuclei per sample.
Then analyze fluorescence of fitsy stain nuclei through the FL one channel set at log scale at 400. This PMN purification method usually provides unstimulated neutrophils with a purity greater than 95%from PMN nuclei are isolated with high yields, isolated to nuclei, can be easily recognized as a different population from intact PMN in the flow cytometer with a dot plot upon stimulation. By cross-linking beta one integrins NF kappa B is translated to the nucleus and this increment in nuclear NF kappa B is detected as an increase in fluorescence.
Similarly, stimulation of PMN by cross-linking beta two integrins also induces NF kappa B activation as indicated by an increase in fluorescence. The sensitivity of this method allows detection of small changes in nuclear factor levels as evidenced by the fact that beta one integrins, which bind extracellular matrix proteins such as fibronectin induce stronger NF kappa B activation than beta two integrins, which bind to adhesion molecules on other cells. This method presents great flexibility because many different fluorochromes can be used and because it allows preparation of nuclei from different cell types, used this simple and economical approach for signal transduction studies that involve changes of protein levels in the nucleus of a variety of cell types.
