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- LITERATURE CITED
FCM is a generally accepted tool to analyze apoptosis. Unfortunately, the cell preparation of all commercial kits available includes cell washing known to cause cell loss which is most likely to affect apoptotic cells in particular. To address this, we developed a seven-color single-platform no-wash analysis technique and compared the results with those from an analogous procedure including cell washing. A five-color mAb cocktail was employed to address target cells by surface labeling, Yo-PRO-1® and DAPI were used to discriminate apoptotic and necrotic from viable cells. Cells were quantified on the basis of internal-standard fluorescent beads. Jurkat cells ACC 282 treated with camptothecin were employed to establish the staining procedure, which was then applied to blood cells collected by extracorporeal apheresis and treated with UV irradiation. Data evaluation showed that although each method by itself was highly reproducible (R2 = 0.973), the numbers of apoptotic cells detected with the no-wash procedure were significantly higher than those obtained after cell washing (P = 6.6 E−5, Wilcoxon Test). In addition, the observed differences increased with higher cell numbers (Bland and Altmann). We conclude that the described test is a feasible and reliable tool for apoptosis measurement and it provides results that are definitely closer to the truth than those obtained from kits that require cell washing. © 2010 International Society for Advancement of Cytometry
Apoptosis plays a major role in various physiological and pathological processes. Cell demise has been investigated in different fields, also including extracorporeal photopheresis (ECP), a therapeutic intervention used in several auto immune disorders, diseases with pathogenic T cell involvement, and chronic graft-versus-host disease (GvHD) (1). Collected cells are reinfused after in vitro exposure to 8-methoxypsoralen and ultraviolet A (UVA) light. One known effect of ECP on PBMC is the induction of apoptosis (2, 3), which can be analyzed by FCM, a generally accepted and reliable tool for detection of apoptosis on single-cell level (4, 5). Several assays for FCM analysis of different stages of programmed cell death are commercially available but all of them include at least one centrifugation step. Hypothesizing that, after apoptosis-inducing treatment, part of the PBMC will be disrupted by centrifugation and thus will not be present for FCM analysis, our intention was to establish a single-platform, no-wash, and multicolor FCM methodology to quantify apoptotic T cells in ECP products.
To discriminate viable from early and late apoptotic/necrotic cells, we tested several markers. We favored the YO-PRO-1®/DAPI combination which allows discrimination of apoptotic from necrotic cells. YO-PRO-1, a DNA-intercalating dye excited at 488 nm, is a reliable marker for FCM analysis of apoptosis (6, 7). DAPI, a nuclear dye excited at 405 nm, which is commonly used in FCM (8), was applied to address late apoptotic/necrotic cells. Camptothecin treatment of Jurkat cells, a popular concept for inducing apoptosis (9, 10), was employed to establish the methodology.
The aim of this study was to identify and quantify apoptotic T cells in ECP products. No cell washing was performed during the whole period, from apheresis over cell culture until cell analysis. In contrast, cell washing was performed with one centrifugation step just before cell staining. Therefore, the comparison between wash and no-wash procedures on each ECP sample was analyzed. Afterwards, the numbers of cells undergoing apoptosis were calculated as cells per μl using Trucount™ beads (11, 12) as internal standard.
We demonstrate that the number of apoptotic cells per μl is clearly influenced by centrifugation both in the Jurkat cell line and in ECP cell samples.
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We present a new single-platform procedure to quantify apoptotic cells by multiparameter FCM. Many different markers to detect distinct time points of apoptotic pathways are available for detection of apoptosis (14, 15). However, all of the methodologies offered on the market include at least one step of cell washing which, according to our experience, is not in line with quantitative cell analysis. Our assay does not use any cell washing step. Results are thus obtained within a shorter time, and they are very likely to be more accurate. The latter is concluded from our comparison with a cell washing procedure which yields significantly lower results with regard to apoptotic cells (Figs. 2A and 2C).
Using the conventional analysis methodology that includes cell washing, the results are expressed as percentage values. It is generally accepted that cell washing coincides with cell loss, and we assume that this loss is higher for apoptotic than for vital cells. This is supported by our finding that the differences between percentage values are clearly smaller than those between absolute values for apoptotic cells (Fig. 2).
Several Authors used the SP methodology to quantify apoptotic cells. In contrast to our approach, however, they applied cell washing steps at various time points during cell culture or preparation (6, 16, 17) or rinsed cells with PBS after induction of apoptosis (13). Our intention was to demonstrate that in particular apoptotic cells are lost by cell washing.
When starting to establish a single-platform procedure by adapting one of the existing kits, we had to exclude some of them, like the Caspase assay that requires intracellular staining and thus cell washing, or the Annexin V assay which depends on a Ca2+ environment known to interfere with EDTA anticoagulation.
As an alternate to Annexin V, a marker for the phosphatidylserines exposed during the early stages of the apoptotic process (18), we tested YO-PRO-1, another marker for early apoptosis (19) which emits light over the 530/30 nm band pass filter to the FITC channel. To address late apoptotic/necrotic cells, we employed DAPI excited with a violet laser and with a light emission of 450 nm. The advantage of this choice is that there is significant spectral overlap only to the second violet channel (530/30 long-pass filter). In contrast to other dyes like PI or 7AAD, the channels used for the blue and red lasers remain free and can be used for multicolor mAb applications. In our hands, discrimination between live, apoptotic, and late apoptotic/necrotic cells could be easily performed and yielded reliable and reproducible results for both, cell lines and primary human cells. Since there is an obvious need for more complex analysis procedures for different types of cells and a broader fluorochrome panel (20, 21), our approach supports the demand for an extended flow cytometric application.
To obtain PBMC, it is common practice to perform density gradients which require several centrifugation steps. Since we aimed to detect apoptotic T cells in ECP products on consecutive days, but wanted to omit centrifugation, we set up a whole-buffy-culture immediately after apheresis. This approach for culture of PBMC worked very well, because the nucleated cell number in the original products was sufficiently high (8–13 × 106/ml). Because of the relatively low number of red blood cells, erythrocyte lysis reported to induce apoptosis in lymphocytes (22) was not necessary in any of the experiments.
In the described multicolor staining for ECP products, we addressed T lymphocytes using CD3 and employed other markers to define NK cells, B cells, and monocytes. It is known that due to membrane loss several leucocytes downregulate surface lineage antigens with beginning apoptosis (23). This was not the case for CD3 which remained stably expressed (not depicted) and therefore served as a reliable marker also for apoptotic T cells, which showed decreasing FSC signals during demise (24).
Our present data strongly indicate that cells, particularly those in apoptotic degradation, are disrupted and lost during centrifugation. Certainly this is of great importance when dealing with samples from cell culture (both primary cells and cell lines). False results from apoptotic cell enumeration could not only influence subsequent experiments but might also have a negative impact if used for therapeutic treatment of patients by ECP.
In conclusion, the presented no-wash/single-platform flow cytometric approach to quantify apoptotic T cells yields reliable and reproducible results which differ significantly from those obtained after cell washing. On the basis of this knowledge, experiments that aim at the detection of apoptotic cells should be no longer performed with a method comprising cell washing, if quantitative results are important. As outlined, the single platform method is feasible and reliable and not only applicable for fresh blood but also for cultured cells.