Supplementary Materialscells-04-00040-s001. conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell features was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the degree of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements carried out only prior to the start of the assay. have suggested that acceptance criteria for a healthy PBMC sample should have a viability 89% when tested with Trypan Blue . We, and others, have mentioned that Trypan Blue is not ideal for measurement of cell viability due to staining artifacts , large numbers of false positive lifeless cells resulting from cells having a reversible damage of their cell membrane , and false negatives from cells that have already initiated the apoptotic pathway but still possess intact cell membranes. On the other hand, Acridine Orange and Propidium Iodide staining offers been shown to be a more accurate means for detecting live and lifeless cells, respectively . Several Rabbit polyclonal to ARC methods are used to determine apoptotic cells. One common method is to detect the flipping of Phosphatidylserine (PS) in the cell membrane by Annexin binding. Since PS flipping is definitely potentially reversible, Annexin staining is not a definite marker for apoptosis . The Yo-Pro family of dyes is also commonly used for detecting apoptotic cells. These are monomeric cyanine dyes that bind to nucleic acids of cells. Since normally, these dyes are impermeable to cell membranes, they bind to DNA in apoptotic cells with jeopardized cell membranes. The Yo-Pro family of dyes functions inside a Calcium-independent, non-reversible manner  and therefore is definitely a more accurate marker for apoptosis. Among the various acceptance criteria for PBMC, measurement of the numbers of apoptotic cells prior to performing a cellular assay has been established as the most accurate. Inside a landmark publication, the acceptance criteria for PBMC were suggested to be 89% viable cells with the portion of apoptotic cells not exceeding 18% . In this study, we display that mere measurements of live/lifeless ratios and apoptotic cell frequencies prior to seeding the PBMC into a T cell assay are not necessarily reliable markers for PBMC features. Measuring the apoptotic cell portion at the beginning and at Sodium Danshensu the end of the assay, however, was Sodium Danshensu found to be a more reliable marker to detect damage to PBMC and therefore their practical impairment. With this study we also resolved the query of whether the presence of apoptotic bystander cells affects T cell features. Apoptotic cells are known to send complex signals to macrophages, entailing find me, eat me, and don’t eat me communications Sodium Danshensu that direct the clearance of apoptotic cells while avoiding pro-inflammatory reactions from the phogocytosing macrophages. The second Sodium Danshensu option protects healthy bystander cells from becoming damaged . Some of the relevant signaling molecules are found within the cell surface of apoptotic cells such as Phosphatidylserine  or ICAM-3 . A change in cell surface charge is also perceived by macrophages as an indication of apoptosis . Other signaling molecules are secreted by apoptotic cells acting as chemotractors to macrophages. They include Lysophosphatidylcholine (LPC) , Annexin-1 , Fractalkine , and Lactoferrin . On the other hand macrophages, upon apoptotic cell engulfment, secrete anti-inflammatory cytokines such as TGF- and IL-10 [26,27]. Since all these processes could potentially impact T cell activation and function, we tested whether the presence of apoptotic bystander cells present PBMC would impact the results of T cell ELISPOT assays. 2. Experimental Section 2.1. Thawing and Handling of PBMC Cryopreserved PBMC.