While many of the stages of the cell-mediated immune response are known, the mechanisms by which various white blood cells can specifically bind (and release) to initiation proteins is not as well known. Some of the challenges in understanding are due to the short time period of this binding and initiation, as well as the short lengths of proteins involved in the process, which can make it difficult to detect the non-polar and polar domains involved in specific binding.
To gain a better understanding of this adhesion process, Researcher Michael Hill, Ph.D. at the State University of New York at Buffalo (UB) used surface plasmon resonance (SPR) with Reichert’s SR7500DC, 2-channel instrument to study the activation and binding properties of HL-60 (Human promyelocytic leukemia) cells, which have long been used to investigate white cell proliferation and differentiation. This work was also highlighted in a webinar sponsored by Reichert and hosted by LabRoots. (You can view the recorded webinar here).
Traditionally, surface plasmon resonance (SPR) has been primarily used in probing interactions between molecules such as proteins, nucleic acids, carbohydrates and small molecules but has not been commonly applied to investigate whole cells. There are a couple of key challenges to consider when studying whole cells with SPR. First, there is a distance dependency to the SPR signal as sensitivity decreases significantly when exceeding 300 nm from the gold surface and, given that the size of cells are on the micron scale, only the part of the cells that is in this detection range is sensed. This leads to much smaller responses than one would initially expect given the large size of cells. Another major obstacle is that the fluidics in flow-based system can easily clog when running samples with particulates like whole cells creating instrument problems and unusable data.
In addition to excelling at measuring molecular interactions, Reichert’s SPR systems overcome these aforementioned hurdles when examining whole cells. Reichert’s SPR systems are extremely sensitive and can resolve responses that are very low (1 RU) so quality sensorgrams can still be obtained despite low signals from whole cell binding. In addition, Reichert’s SPR systems implement a very robust fluidics platform that can accommodate a variety of sample compositions, such as cells, without the risk of clogging.
The cell study performed by UB researcher Michael Hill shows that Reichert’s SPR systems can provide valuable information on the specificity of white cell binding during the initiation of an inflammatory response, even when studying this interaction across multiple protein layers. This research focuses on the specific interaction between immobilized P-selectin and white blood cells (HL-60). During inflammation, white cells are recruited to the injury site by selectins that are released by endothelial cells. Selectins tether to white blood cells and thereby determine the rate of recruitment at the injury site. The process is illustrated by the Figure below:
By running control experiments with SPR, this work shows that specific adhesion of P-selectin to HL-60 can be probed when a blanket of albumin (Bovine Serum Albumin) is used to block non-specific adhesion. Reichert’s SPR systems can identify specific binding across multiple protein layers, and can monitor how the cells spread over the surface bearing ligands, even over narrow distances. “This is not possible with light microscopy,” Dr. Hill notes. He adds that he hopes future experiments can visualize the interaction as it is being measured by SPR.
The Figure below presents the results from Researcher Hills’s SPR experiments. BSA blocking ensured that specific interactions were probed and the monoclonal antibody (MAb) KPL-1 (anti-PSGL-1 mab) confirmed the specificity of these interactions.
This work shows that Reichert’s SPR systems can be extremely useful for probing specific and non-specific cell adhesion, which can often exist at the same time in certain biological situations. SPR in this case was also helpful for examining characteristics of cell adhesion in very short time periods and involving very short lengths of interacting protein. Further, Dr. Hill reported that using Reichert’s SR7500DC SPR system allowed for quantitative results on the strength of cellular interactions with proteins, and that the technique was cheaper, simpler and easier than other methods such as atomic force microscopy.