Study On Fluorescence Dye CFDA-SE Labeling For Assay Of Lymphocytes Proliferation And Cell-mediated Cytotoxicity

Analysis of proliferation and cytotoxicity of lymphocytes in vitro is an important parameters for the estimate of body immune function, study of disease mechanism, vaccine of anti-tumor and drug. So the assay for division and proliferation of specific cell and its subset on a single cell level and the analysis for kinetics of immune responses and the determination cell cycle is significant on investigating the phenotype and marker which are necessary for the cell differentiation and the proteins which are involved in the process of cell division. CTL and NK cells play the most important roles on cell-mediated cytotoxi- city, so assay the activity of CTL and NK is a crucial indicator of the immune defense against tumor or virus. Thus, it is mostly important how to evaluate the function of effector cell and quantitate the lysis percentage precisely and the apoptotic pathway of target cell.Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA-SE) is a membrane permanent fluorescence dye. CFDA-SE binding is stable and nontoxic to cells. It is suitable for long-term incubation experiment.The purpose of our study is to determinate the proliferation of lymphocyte and cell-mediated cytotoxicity by the combination of CFDA-SE labeling and flow cytometry. The principal of analysis of lymphocyte proliferation is: CFSE is divided equally into daughter cells following cells division and its fluorescence intensity is the half of the parental generation. So in a whole cell population which undergoing proliferation, the fluorescence intensity declines in half with the following generation proliferated. We can decide the level of proliferation and how many generations proliferated by the FL1 channel of flow cytometry on a single cell level. The principal of analysis of cytotoxicity is: firstly the target cells are labeled by CFSE with the purpose of discriminate from effect cells. Secondly the target cells with damaged membrane are labeled by propidium iodide(PI)which is the DNA-combined dye. The double positive cells which are labeled by CFSE/PI are counted by FCM and then the percentage of dead cell are calculated on a single cell level.Assay for lymphocyte proliferation by CFDA-SE labelingWe prepared the lymph-node cells,thymocytes,splenic mononuclear cells of BalB/C under sterilely condition,and stained with different concentration of CFSE and then analyzed the intensity of fluorescence by fluo-microscope and FCM. The proliferation of PBMC labeled by CFSE or unlabeled by CFSE and stimulated by ConA can be analyzed by 3H-TdR method to investigate the effect of CFSE on the ability of proliferation of lymphocyte and the optimal labeling concentration of CFSE. PBMC and thymocytes are labeled with the optimal concentration of CFSE and cultured on the 24 well plates which contains IL-2 culture medium and proliferate under the stimuli of ConA,anti-TCRαβmAbs,anti-TCRαβ+ CD28mAbs and PMA+Ion, the cell proliferation can be estimated by FCM CellQuest acquire/analyze software. The ability of proliferation of susets of PBMC T cell can be analyzed by ModFit TM V3. 0 and then compare percentage of different cell cycle,PI and PF. T,B cell in mouse splenic mononuclear cells show different ability of proliferation under the stimuli of anti-CD3 mAbs and LPS singly or in combination and can be further verified by FCM. We can also analyze the proliferation of PBMC and CIK induced by PHA by the same method.The results show: the lymphocyte from PBMC and ;thymus T cells can be activated and differentiate by different stimuli and IL-2. Among them, PMA Ion is the strongest stimuli, TCRαβ+CD28mAbs are the second, ConA is the third. TCR mAb stay the at last position。The result of the lymphocyte of PBMC has stronger ability of proliferation than immature thymus T cells comes from the data of generation of cell differentiation and percentage of cells. The proliferation kinetics of subset of T cell stimulated by ConA shows:CD4 + and CD8 + T are different in terms of speed of differentiation after culture for 24 h, this phenomenon is much more obvious after 72h. The percentage of parental cells for CD4+T is much higher than CD8+T for the third generation while is much lower than CD8+T for the fourth and sixth generation. The corresponding result for CD3+T is between CD4+T and CD8+T. B cell shows high ability of proliferation when differentiate into the seventh generation under the co-stimuli of LPS and anti-CD3mAbs. The result of FCM shows:the proliferation of CD8+ prior to CD4+T when we treat PBMC with PHA. The percentage of CD3+CD56+T in the population of PBMC increases from 5.39% to 83.91% after induction of CIK for 7d. the cells of 97.13% differentiated and peaks at the 6～7 generation。In conclusion:the combination of CFSE labeling and flow cytometry can analyze the proliferation and differentiation of lymphocytes and their sub clones on single cell level and then indicate the progress of immune proliferation kinetically.Cell labelingFour tumor cell lines were labeled with CFDA-SE and subjected to analysis by a flow cytometer. These were the human erythroleukaemic cell line K562, mouse lymphoma cell line YAC-1, human mammary cancer cell line MCF-7 and human melanoma cell line A375. We assay the fluorescence from 0 to 24h kinetically and determine the optimal concentration for labeling by CFDA-SE. The incubation period for CFSE differs from 1h to 6h and then is labeled by PI and analyzed the percentage of apoptotic cell, namely spontaneous death which means the toxicity of CFSE. We incubate cell with CFSE from 5 to 15 min and test the intensity of fluorescence and spontaneous cell death to determine the optimal labeling time by CFSE.The results show: All groups of MFI are lower than that of 0h (the process of equviation). MFI declines with the incubation of CFSE and tends to stabilizing at 4h. the MFI increase with the concentration of CFSE when we label the four tumor cell lines with 1μmol～10μmol CFSE. The MFI among the four cell lines are different when we label them at the same concentration of CFSE. The intensity of fluorescence in the histogram was showed Gaussian distribution,we select the one which is beyond 2.0 for MFI as the optimal labeling concentration. The optimal concentration for K562 and YAC-1 is 2.5μmol,while the optimal one for A375 and MCF7 are 5μmol and10μmol respectively. We label cell with1μmol～10μmol CFSE and culture for 4h and then stained by PI, the spontaneous cell death shows no change with the increasing of concentration of CFSE and no significant difference(P>0.05) with the mock labeling cell. The labeling periods are 5 min,6 min,7 min,8min,10min,15min. We assay the MFI and cell death with the different labeling periods after incubation for 4h. The optimal labeling time is 8min and MFI >2 which means we can between CFSE negative and positive cells on this condition. The percentages of cell death for all groups of labeling period are below 5%.In conclusion, the optimal labeling concentration of CFSE for different cell lines is different. CFSE is not toxic for cell and the spontaneous cell death is below 5%. So CFSE is a promising fluorescence dye for cell labeing.Assay for cytotoxicity by the combination CFSE/ PI labeling with cytometricWe label the target cell K562,Yac-1 with CFSE and incubate them with human PBMC,CIK and spleen PBMC from BalB/C mice. The ratio of effect cell and target cell is from 100:1 to6.25:1,the incubation period is from1h to 6h and label the damaged target cell with PI. We assay the CFSE/PI positive cells with flow cytometry and calculate the percentage of cell death for target cells to estamite the cytotoxicity of the three effect cell lines. We compare the cytotoxic effect of CIK among the four target cell lines which are K562,HL- 60,Hela,MCF7,A375 and the synergistic killing effect of NK sensitive(K562) and non-sensitive (MCF7)target cell line with the combination of anti- CD45mAbs. we also investigate the apoptotic pathway mediated by CIK with Annexin V /PI staining. We compare the sensitivity between the flow cytometry combined with CFSE/PI and 51Cr releasing method by setting up a group of killing tests in which the ratios of E/T are 25:1,12.5:1,6.25:1,3.125:1 and the incubation period is 4h, while the other group in which the incubation periods are 0.5h,1h,2h,4h and the ratio of E/T is 25:1. in order to evaluate the influence of the population of cell on the result, the ratio of E/T is 50:1, we compare the samples for which the effect cell are 5×105,2.5×105, 1.25×10~5 respectively;we collect 500,1000,2000 target cells for each sample and calculate the average CV value and evaluate the influence of the minimal collected target cell on the result with the condition of on effect on the sensitivity of the experiment.The results show: the percentage of specific cytotoxic activity increase with the incubation period when the ratio of E/T is 25:1,12.5:1,6.25:1,the percentage of specific cytotoxic activity tends to stabilize at 2～4h and peaks at 4h and declines at 6h when the ratio of E/T is 100:1 and 50:1. the optimal incubation period is from 2h to 4h. CIK shows higher cytotoxic effect for K562, HL-60 and Hela with the results are 83.52%,60.15%,57.32%respectively. However, CIK shows lower cytotoxic effect for MCF7 and A375 with the results are approximately 20%. The different cytotoxic effect maybe come from the fact that both MCF7 and A375 are adherent and then can not contact with the effect cells effectively. The CD45vsPI biparameters histogram shows: the percentage of cytotoxicity of CIK for K562(CD45+PI+)is 34.64% and for MCF-7(CD45-PI+)is 14.92%.the result of apoptosis of K562 shows that the percentage of AnnexinⅤ(71.75%)is higer that that of PI(59.25%) which can be explained by the fact that the convert of PS from inside to outside of the membrane is earlier fact than the increase of permiability of the memberane and then PI can goes into it. So we can conclude one of the mechanisms by which CIK kill K562 is membrane mediated apoptosis. We can conclude by comparing the sensitivity of methods that the percentage of cell death is 21.45% assayed by FCM when the ratio of E/T is 3:1 while there is almost no cell lysis assayed by the 51Cr releasing method. The percentage of cell death is 20.47% assayed by FCM after incubation of 0.5h while it is only 3.15% assayed by the 51Cr releasing method. In order to reduce the amount of effect cells,we compare the killing effect with different effect cells which are 5×105,2.5×105 and 1.25×105 respectively and the ratio of E/T is 50:1,the result shows that the percentage of killing of different groups are similar with the average value are 36,35 and 35.5%。when we collect 500,1000,2000 for each group, the average CV are 22,24.5,24 and 24.5 and the S.D values are similar which means how many target cells we plan to collect does not affect the result.In conclusion:the combination CFSE/PI labeling with flow cytomery can analyze cell-mediated cytotoxicity on a single cell level. The additional combination with specific antibody can analyze target cell on multi-parameter level. The method we focused in this paper is much more sensitive than the 51Cr release assay and is useful for setting of lower ratio of Effector/target cell and incubation period. It only need small amout of effector cells and we can obtain reproducible and comparable results.