The cytometry and biomarkers technology unit supports biomedical projects through state-of-the-art technologies and pipelines for cell phenotyping and molecular profiling with single cell resolution. In this context, we use different tools for single cell multiomic analysis to obtain insights in into gene expression and gene regulation in health and disease. Flow cytometry identify and source individual cells based on unique characteristic.
Recent advance include eye throughput, image-based spectral sorters, and those technology offer flexibility in panel design enabled simultaneous use of similar dyes and the managing of autofluorescence. A major experimental challenge when using cutting edge single cell technologies is working with particularly fragile or rare cell populations. Each tissue or cell type needs to undergo fine tuned protocol optimization to obtain enough highly viable cells that translate into high quality sequencing data.
Our modified single nuclei preparation protocol improves nuclei recovery and minimize the stress on the nuclei. It's specifically optimized for brain and bone marrow tissues and covers all the steps from sample association to nuclei purification. To begin, fill a glass dounce with two milliliters of ice cold nuclei lysis buffer containing 0.01%digitonin and add the dissected mouse brain tissue pieces into it.
Using a glass dounce tissue homogenizer, homogenize the tissue 25 times each with pestles A and B and transfer the resulting homogenate into a 15 milliliter tube. Add two milliliters of ice cold nuclei lysis buffer containing 0.01%digitonin to the homogenate and incubate on ice for five minutes. Then centrifuge the nuclei at 500 G for five minutes at four degrees Celsius.
Using a micro pipette, remove the supernatant and add four milliliters of ice cold nuclei lysis buffer containing 0.01%digitonin. Filter the nuclei suspension through a 40 micrometer cell strainer. Centrifuge the nuclei again, remove the supernatant and add four milliliters of staining buffer to wash the nuclei.
After filtering the suspension through a 40 micrometer cell strainer, pellet the nuclei by centrifugation and resuspend the nuclei in one milliliter of PBS, containing 0.04%BSA and RNA inhibitors. To count the cells, mix 10 microliters of 0.4%trypan blue with 10 microliters of the nuclei in a 0.5 milliliter tube. Count the nuclei using an automated cell counter.
Transfer 100 microliters of the nuclei to a facs tube for the unstained control. Add 10 microliters of 7AAD to the remaining nuclei and incubate for five minutes at four degrees Celsius and proceed with nuclei sorting using facs. Then transfer 10 microliters of the sorted nuclei into a new facs tube containing 90 microliters of DPBS with 2%heat inactivated FBS.
After centrifuging the sorted nuclei, remove the supernatant using a micro pipette and resuspend the nuclei in 100 microliters of diluted nuclei buffer. Before sorting, the sample contains substantial debris with over 99%of singlets positive for nuclear stain indicating effective cell lysis. Brain nuclei after sorting demonstrated an increase in purity from the initial 36%to nearly 100%To begin, take a vial containing the mouse bone marrow hematopoietic stem and progenitor cells.
Using a micro pipette, resuspend the pellet in one milliliter of a ACK lysine buffer and incubate for one to two minutes at room temperature. Transfer the resuspended mixture into a 50 milliliter tube through the pre-wetted 70 micrometer cell strainer. Then add 10 milliliters of facs buffer to dilute the A CK lysine buffer.
Centrifuge the mixture at 400 G for five minutes at four degrees Celsius, and resuspend the pellet in 10 milliliters of facs buffer by first resuspending it in one milliliter of buffer and then topping up with nine milliliters of buffer. Now mix 10 microliters of 0.4%trypan blue with 10 microliters of the cells in a 0.5 milliliter tube, and count the cells using an automated cell counter. To stain the cells, centrifuge the cells at 400 G for five minutes at four degrees Celsius.
Resuspend the pellet in facs buffer to achieve a final concentration of one times 10 to the seven cells per milliliter by first resuspending the pellet in one milliliter of buffer and then topping up with the remaining volume. Using a P1000 micro pipette, transfer the suspension into a facs tube through a 35 micrometer cell strainer cap. Next, prepare staining mix as shown here.
After centrifugation, resuspend the cell suspension in 300 microliters of staining mix one in a sample tube and incubate on ice for 15 minutes protected from light. Then add 300 microliters of mix two into the sample tube and incubate for 20 minutes on ice. Protected from light.
Add three milliliters of the facs buffer to the single stained and mixed stained sample tubes. Now centrifuge the cell suspension. After discarding the supernatants, resuspend the pellet in 500 microliters of the facs buffer.
Prepare a 1.5 milliliter tube prefilled with 500 microliters of collection medium. Once the facs instrument has been calibrated, add 500 microliters of facs buffer to the cell suspension and then transfer one milliliter of the sample through a 35 micrometer cell strainer cap into a new facs tube. After cell sorting, transfer 10 microliters of the sorted cells into a new facs tube containing 90 microliters of facs buffer.
Pellet the cell suspension by centrifugation and resuspend in 50 microliters of PBS with 0.04%BSA. Transfer the suspension to a 0.2 milliliter tube and add 50 microliters of PBS with BSA to the original tube to collect any leftover cells. Transfer the cells to the 0.2 milliliter tube to reach a total volume of 100 microliters.
Run a pilot experiment to identify the best lysis time for nuclei isolation. After pelleting the nuclei, resuspend the nuclei pellet in 12 microliters of diluted nuclei buffer. Add two microliters of nuclei to a tube containing 0.4%trypan blue and eight microliters of PBS with 0.04%BSA.
And count the nuclei using an automated cell counter. After completing the nuclei isolation, transfer six microliters of nuclei resuspension into a new facs tube pre-filled with 150 microliters of facs buffer. Add three microliters of 7AAD and incubate for five minutes on ice.
