In order to study the chromatin DNA interactions that govern neuronal specific gene expression in the human brain, a pure population of neuronal nuclei must first be obtained to do this. Postmortem human brain tissue is homogenized and the total nuclei are isolated using density gradient centrifugation. Then nuclei are labeled with neuronal specific antibodies isolated via flow metric sorting, and finally concentrated with ultracentrifugation.
Hi, I'm Anish Matan from the laboratory of Dr.Sharon mc Barian here in the psychiatry department of University of Massachusetts Medical School. Today I will show you a procedure for neuronal nuclei isolation from postmortem human brain tissue. So let's get started.
For each nuclei sample to be sorted, start with a thousand milligrams of frozen postmortal brain tissue. With this amount, you can obtain about 5 million neuronal nuclei after fluorescence activated sorting. To begin the nuclei extraction, place 250 milligrams of brain tissue into a down downer containing five mils of lysis buffer, and place it on ice.
Once the tissue thaws down it for one minute while on ice. After downing, transfer the homogenized tissue to a 15 mil clear ultracentrifuge tube and then put the tube on ice. Repeat this homogenization for the remaining tissue.
Next, carefully pipette nine mils of sucrose solution to the bottom of the ultracentrifuge tubes to set up a concentration gradient such that the homogenized tissue solution is on top of the sucrose solution. Now weight the ultracentrifuge tubes to ensure that they're balanced During the ultracentrifugation. Make any adjustments to the weight by adding lysis buffer to the top layer After the tubes have been weighed, place them in the ultracentrifuge using the SW 28 rotor ultracentrifuge samples at 107163.6 RCF for 2.5 hours at four degrees.
Once the centrifugation is complete, aspirate off the snat as well as the layer of the debris found at the concentration gradient. Be careful not to disturb the pellet at the bottom of the tube, which contains the nuclei. Next, add 500 microliters of one XPBS to each pellet and let it sit on ice for 20 minutes.
After 20 minutes, resus suspend the pellets by pipetting up and down. The incubation on ice allows the pellets to dissolve a little on their own, which makes it easier to resuspend the nuclei, thus lessening the amount of stress the nuclei experience. Then transfer the resus suspend and nuclei to two mil centrifuge tubes.
Combine the nucleic solution from two of the ultra centage tubes to each micro centrifi tube and again, place the samples on ice. Now that the nucleic extraction is complete, I'm going to move on to the immuno staining step in order to isolate neuronal nuclei by fact, sorting the sample containing nuclei will be incubated with an immuno staining mixture composed of primary and secondary antibody, as well as blocking links to make the immuno staining mixture at 1.2 microliters of neuronal specific antibody new N to 300 microliters of one XPBS. Then add a hundred microliters of blocking mix, which contains 0.5%BS, A, and 10%normal code serum in one XPBS.
Finally, add one microliter of the secondary antibody called LOR 4 88. Also prepare an immuno stain for a negative control sample for this immuno stain mixture. Exclude the primary antibody new end and replace it with 1.2 microliters of one XPBS.
Now vortex immunostaining mixtures and incubate them for five minutes at room temperature in the dark while the staining mixtures are incubating. Prepare the control nuclei samples for the negative control sample Eloqua 20 microliters of the resus span nuclei into a two mil tube containing 980 microliters of one XPBS. Also prepare an unstained control, which contains the nuclei solution and PBS only.
Now Eloqua 20 microliters of the nuclei into another tube and raise the volume back up to one mil with one XPBS. Then place all samples back on ice. By now the immuno staining mixtures are finished.
Incubating add 401 microliters of the mixture into the appropriate sample. The nuclei sample received the mixture containing both new and and LOR 4 88. The negative control sample received a mixture containing only the secondary antibody LOR 4 88 while no immuno stain is added to the unstained control sample.
Next, transfer the samples to the coldron and rotate them in the dark for 45 minutes. Now that the 45 minute incubation is complete, I will walk the samples over to the University of Massachusetts Medical School flow cytometry core facility to do some fluorescence activated sorting During the transport to the core facility, the samples should be kept on ice and in the dark. At the flow cytometry core facility, the samples are first filtered through a 40 micron filter and then they're run through the instrument for a few minutes while being kept cold.
This is done in order to get some preliminary data before sorting. The unstained control establishes the degree of negative fluorescence. Based on this information, adjust the machine sensitivity to fluorescence.
The stained control is used to control flu. Any background noise due to out of fluorescence at this point set the appropriate gates required for sorting. Several different gates are used to sort samples.
Here's some typical data for the first gate. This fax analysis chart shows a representative sample after the gate has been adjusted. The first gate recognizes patterns of debris and viable nuclei based on a combination of settings such as fluorescence and general visual experience.
The x axis represent forward scatter, which corresponds to relative size while the Y axis account for site scatter, which accounts for internal complexity such as granularity. The first gate can also give an idea of the size of the different nuclei found within our sample. This allows to separate the debris from the actual nuclei, and here's some typical data from the second gate.
This fax chart shows an example of a second gate, which allows to sort nuclei individually while discarding any aggregates. This gate looks at differences in lengths of time that nuclei take in order to go through the laser. Those which are aggregates normally take longer to pass through.
This particular parameter is very important as inclusion of aggregates can interfere with purity since some fluorescent nucle may be bound to non fluorescent ones. Finally, here's typical data from the third gate. This gate is set to a specific fluorescence wavelength matching that of the fluoron found in our secondary antibody.
In this fax chart, you see the fluorescence level along the x axis and a deep red channel detector, PECY seven along the Y axis. These dot blots give a better visual when simply compared to a single color histogram. Placing SCADA allows to sort and separate new and positive neuronal nuclei from new and negative non neuronal nuclei.
After all the gates are chosen, the nuclei are sorted into one XPBS. Once the whole sample has gone through the fact sort, check the purity of the sort by running an Eloqua of the sorted sample through the instrument. Here's an example of an optimal result.
Here compare the presort sample, which contains a mixed population of the nuclei to the sorted Eloqua that has eliminated most of the negative nuclei. Note that purity percentages are based on the number of events that contaminate the sample based on the gates that were set. Keep in mind that the first time the sample is run through the machine, some bleaching may occur, thus causing some dimming to account for this, some adjustments may need to be made.
Once the sample has been sorted, begin processing the nuclei. To do this, take 10 mils of the sorted sample and add two mils of sucrose solution, 50 microliters of one molar calcium chloride, and 30 microliters of one molar magnesium acetate. If there's less than 10 mils of sorted sample, raise the volume up to 10 mils by adding one XPBS or adjust the additional materials accordingly.
Next, invert the sample several times and place it on ice for 15 minutes. After 15 minutes, centrif feature sample in a swing bucket rotor 1, 786 RCF for 15 minutes at four degrees. After centrifugation carefully aspirate off the supernatant without disturbing the white pellet found at the bottom of the tube.
Then dissolve the pellet in 200 microliters of the appropriate solution needed for the preceding experiments. In this case, we're dissolving the pellet and downing buffer for subsequent chip experiment. Then resuspend the pellet by pipe up and down.
At this point, you can transfer the nuclei into a smaller tube and place it into a minus 80 degree Celsius freezer. Until further use, well, I've just shown you how to isolate neuronal nuclei from human postmortem brain tissue. With this nuclei, you can carry out a number of different analyses such as chromatin immunoprecipitation.
When doing this procedure, it is important to remember to always keep your samples on ice since the nuclear are unfixed and can degrade at long temperature. Also, it is important to handle human tissue at biosafety level two or higher safety standards. So that's it.
Thanks for watching and good luck with your experiments.
