The overall goal of this procedure is to isolate subsets of precursor B cells from umbilical cord blood. This is accomplished by first isolating mononuclear cells from cord blood by density gradient centrifugation. In the second step cell surface antigens are labeled with biotin conjugated antibodies to magnetically deplete any of the remaining non B cells.
Next, the isolated B cells are fluorescently labeled with antibodies against the CD 19, CD 34, and CD 45 cell surface antigens. In the final step, the cells are sorted by flow cytometry to recover the precursor B-cell subsets. Ultimately, cells of sufficient number and quality can be acquired for utilization in downstream assays requiring high quality DNA and RNA.
One advantage of the cell sorting strategy over alternative methods is that our strategy requires less time on the flow cytometer and only three fluorescent antibodies greatly reducing the cost of the experiment. Generally, individuals new to this method will struggle with the variety of methods available for preparing umbilical cord blood cells for flow cytometry and the complexity of the preparation of the flow Cytometer. Though this method can provide insight into healthy precursor B cells, it can also be applied to other cell types present in umbilical cord blood such as T cells or to studies of diseases involving any hematopoietic cells such as leukemia or lymphoma.
Visual demonstration of this method is critical because it is difficult to describe the proper techniques required for processing the umbilical cord blood and cells in a way that maximize the cell viability and reduces the presence of contaminating debris. To isolate mononuclear cells from umbilical cord blood first dilute eight milliliter aliquots of the cord blood with 24 milliliters of DPBS. Then slowly layer each diluted blood mixture on top of 15 milliliter ALI watts of fipa plus in 50 milliliter conical tubes, taking care to keep the layers separate.
Next, centrifuge the blood for 40 minutes at 400 times G and 20 degrees Celsius while the cells are being separated by density gradient label seven five milliliter round bottom tubes with the sample ID date and any other appropriate information as outlined in the table. After the separation, the mononuclear cells will remain at the plasma fi opaque plus interface, whereas the granulocytes and erythrocytes will sediment now aspirate the upper plasma layers, avoiding contact with the mononuclear cell layers. Then use a 10 milliliter glass pipette to carefully collect the mononuclear cell layers from three of the 50 milliliter tubes into a single new 50 milliliter tube.
Fill this new tube with PBS, mix the cell suspension gently and then wash the cells two times for 10 minutes at 300 times G and 20 degrees Celsius. After the first wash, resuspend the pellets and transfer to a single 50 milliliter conical tube. After the second wash, gently resus suspend the cell pellet in 200 microliters of PBS transfer one microliter of the cell suspension to one milliliter of PBS in a 1.5 milliliter micro centrifuge tube for counting.
Then wash the cells in the 50 milliliter tube with more PBS afterwards completely removing the supernatant without disturbing the pellet. To isolate the B cells from the mononuclear cells first Resus suspend the just washed pellet in 160 microliters of four degrees Celsius. Freshly prepared DGAs E-D-T-A-P-B-S buffer.
Then mix 40 microliters of the B-cell isolation kit, bio ITIN antibody cocktail with the cell suspension. After incubating the cells for 10 minutes at four degrees Celsius, wash away the free antibody in one milliliter of four degrees Celsius, E-D-T-A-P-B-S buffer per 10 million cells. Then resuspend the cell pellet in 320 microliters of four degrees Celsius E-D-T-A-P-B-S buffer.
Now mix 80 microliters of anti biotin microbeads with the cell suspension after a 15 minute incubation at four degrees Celsius. Wash the cells again while the cells are spinning. Place an LS column in the magnetic field of a max separator and drip three milliliters of four degrees Celsius E-D-T-A-P-B-S buffer through the column.
Next resuspend up to 100 million of the washed cells in 500 microliters of four degrees Celsius, E-D-T-A-P-B-S buffer. Then pipette the cell suspension onto the column collecting the unlabeled cells that pass through the column in a 50 milliliter conical tube. Add an additional two milliliters of four degrees Celsius, E-D-T-A-P-B-S buffer to the walls of the original conical tube, one milliliter at a time and then gently mix and pipette the residual cell suspension onto the column each time to be certain to recover all of the cells.
Finally fill the conical tube containing the unlabeled cells with PBS. After centrifuging, the cells carefully remove the supernatant without disturbing the cell pellet. Then gently resus suspend the cells in 200 microliters of E-D-T-A-P-B-S buffer.
Begin the antibody labeling. Step by first transferring one microliter of the cell suspension into the previously labeled unstained tube. Add 500 microliters of E-D-T-A-P-B-S buffer to the tube and store it on ice.
Turn off all lights and then add 20 microliters each of CD 19, CD 34, and CD 45 antibodies per 1 million cells into the remaining cell suspension. Mix well and place the tube in the dark for 30 minutes at room temperature. Next, transfer 40 microliters of the antibody labeled cell suspension into the seven A a D labeled tube.
Wash the cells in one milliliter of PBS and then resuspend the pallet in 100 microliters of binding buffer. Add seven microliters of seven A a d to these cells and then incubate them in the dark for 10 minutes at room temperature while the cells are being labeled with seven A a d top off the tube of the antibody labeled cells with PBS wash the antibody labeled cells and then resuspend the pellet in 500 microliters of E-D-T-A-P-B-S buffer. Now transfer the entire volume of the antibody labeled cell suspension into the CD 19 positive CD 34 positive CD 45 positive labeled tube.
Rinse the tube with an additional one milliliter of E-D-T-A-P-B-S buffer and transfer the residual cell suspension into the CD 19 positive CD 34 positive CD 45 positive labeled tube. Finally, add 300 microliters of binding buffer to the seven A a D tube to sort the cells using the MO flow XDP flow cytometer. First set up the MO flow for sorting, run proper compensation controls and then create a protocol including plots as illustrated in this figure.
Ensure the aerosol evacuation system is running at all times and then use the unstained sample to set the voltage and gain for the forward and side scatter plots. To identify the negative fluorescence populations. Set the threshold to less than or equal to 1%so that any DNA containing debris does not contaminate the recovered samples.
Run about 50, 000 events of the CD 19 positive CD 34 positive CD 45 positive sample. To set the gating strategy as just demonstrated back gating the ungated CD 19 positive CD 34 positive population onto the side scatter versus forward scatter plot. To ensure that the lymphocyte gait includes potential pro B cells.
Use this sample to also set the negative fluorescence population for the seven a a d staining. Next, run the seven a a d sample to determine the sample viability only sort cells from a sample with a greater than or equal to 95%viability at the outset as dead cells will stain indiscriminately and may contaminate the sort populations now set up sort decisions to collect for populations CD 19 positive, CD 34 positive CD 19 positive, CD 34 negative CD 45, low CD 19 positive, CD 34 negative CD 45 medium and CD 19 positive. CD 34 negative CD 45 high.
Include the lymphocyte and doublet discrimination gates in the sort decisions of all the populations to prevent clogs. In the sorting tip, filter the CD 19 positive CD 34 positive CD 45 positive sample through a 40 micron cell strainer immediately before sorting. Then place labeled collection tubes coated with 2%FBS in PBS in an ice packed tube holder and sort the cells into the appropriate collection tube immediately after the sort has completed.
Extract the DNA between sample variation plays a role in the success of the cell, sort in a good sort. There is a high percentage of cells in the lymphocyte gait. Samples with good success rates contain low levels of contaminating debris as evidenced by the low number of events that fall below and to the left of the gated lymphocyte population.
The samples with poor success rates contain high levels of contaminating debris. Poor samples show a marked increase in these events. The flow sorted cells and the subsequent DNA isolated from each precursor B-cell subset are of quantity and quality to perform downstream analyses.
We've routinely utilized this DNA in Myra to enrich from methylated DNA here DNA, isolated from CD 19 positive CD 34 positive cells CD 19 positive, CD 34 negative CD 45 low cells CD 19 positive, CD 34 negative CD 45 medium cells and CD 19 positive CD 34. Negative CD 45 high cells were sonicated on high for a total of nine minutes column purified and then visualized on a 1%aros gel with cyber green nucleic acid gel stain in this lane. A control 100 base pair ladder was also run after Myra using the active motif method collector ultra kit PCR with the control methylated SLC 25 A 37 and control unmethylated APC one genes was performed to confirm the enrichment of the methylated DNA.
The higher amplification of the SLC 25. A 37 confirms the methylated DNA enrichment in the precursor B-cell subsets Once mastered, this technique can be done in 10 hours if it is performed properly. While attempting this procedure, it is important to remember to treat the cell very gently and to use the optimized amount of antibodies so that the generation of contaminating debris is minimized Following this procedure.
Other methods like next generation sequencing can be performed in order to answer additional questions like which genes are methylated and precursor B-cell subsets. Don't forget that working with live human cells can be extremely hazardous, and precautions such as running an aerosol evacuation system should be taken at all times when performing this procedure.
