Cytometry and Antibody Technology

Symptoms: Anything we put through the FACSAria, came out dead. Not like exploded, obliterated dead, just permeable to trypan blue, not-able-to grow, dead. Secondly, tandems looked weird. For example, PE-Cy7 would have a PE-Cy7 positive population, but then it would also have a PE positive population, as if the Cy7 portion of the tandem was getting quenched. However, the PECy7 antibody was fine since it looked normal on the MoFlo.

Troubleshooting: Obviously, the 1st thing to check is the buffer. So, I collected some PBS exiting the Aria’s flow cell, mixed that buffer with some cells, and ran them on the instrument and checked them under the microscope. They were fine, suggesting the buffer itself was not killing the cells. I then turned to the (HPLC) valve that controls sample uptake. I disconnected the sample line, after the valve, but before the flow cell, collected some cells, and then reran them. They were fine again, suggesting that it’s not the HPLC valve that is killing the cells. So, it seems like the cells have to pass through the flow cell in order to die. On the flow cell, there are wires attached to provide the charging of the stream (required for sorting). So, I disconnected all the charging wires and tried again…still dead. Maybe the lasers are frying the cells. Turned the lasers off…still dead. What to do next?

Placing a volt meter on the metal portions of the flow cell, we discovered a 0.5V charge present. So, perhaps if we grounded the flow cell with an external copper wire attached to a grounding source (in this case, an electrical box behind the Aria) we could keep the cells alive…no luck!

Then, it hit me like a ton of bricks. For about a week now, our Millipore Biocel Water Deionizer was down, so I was getting deionized water from the Monoclonal Facility. Now, the monoclonal facility uses this water for all their tissue culture work, so it is definitely a good source of H2O. The output of water was ‘suppose’ to be comparable. But then I thought, if the problem really is an electrostatic charge in the flow cell, and, if the new water source is not deionized to the same degree as our original water source, the electrostatic charge could be passing through the buffer, directly to the cells and basically shocking them. Pretty much like an aggressive electroporation.

All the pieces were coming together. It makes sense. This could also explain the PECy7 issue. The extra electrostatic charge was messing with the fluorescence of the fluorochromes as well. So, the obvious next step was to use water from a Biocel Millipore unit. As soon as I did that, all was fine.

So, in summary, buffer made with water from this specific Millipore system, passing through the flow cell on the Aria, with cells present shocked the cells and killed them. Buffer made with water from the Millipore Biocel system..fine. I never found out from BD if that electrostatic charge is suppose to be there or not, but it seems like properly deionized water is required to protect the cells from this charge.

All is well now. Happy sorting!