It allows to detect protein interactions in all cellular compartments, it is fast and quantitative, non-invasive and highly reproducible. The reason for this might be that an easy to adapt, standardized, well controlled and reliable routine to measure and quantify FRET by FACS is still missing. As controls and to verify our previous results that the assay is sensitive to discriminate co-localization from FRET, we measured FACS-FRET between Rev, the viral transactivator Tat and the multimerization-deficient RevSLT40 mutant. There are some considerations for measuring NKA for diagnostic use in the clinical laboratory: ideally, assays should be non-biohazardous and easy to perform, requiring a small volume of blood. One day post transfection/electroporation FRET-positive cells were sorted in PBS +1% FCS and pelleted. Human PBMCs were isolated from healthy adult donors using density-gradient centrifugation with Ficoll-Hypaque (d = 1.077, LymphoprepTM; Axis-Shield, Oslo, Norway) and washed twice with phosphate-buffered saline (PBS) (Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea). Confocal images were analysed with the FRET and colocalization analyzer ImageJ plug-in . 5A). Alter G, Malenfant JM, Altfeld M. CD107a as a functional marker for the identification of natural killer cell activity. Strikingly, URD expressed together with CD317 showed a strongly diminished FRET signal 14.5% (+/3.8% SD). It is possible that PBMCs contain a relatively higher number of NK cells than WB owing to the isolation process and adjustment of the E:T ratio, which can introduce errors, especially in patients with low or high NK cell counts. (2011) 331:449. the MEM-control with NA7 and mac 239 Nef). Human myelogenous leukemia (K562) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 g/mL streptomycin (Lonza, Walkersville, MD, USA) at 37C in a humidified 5% CO2 incubator.
5). In some experiments, we added coverslips to the wells to analyse subcellular localization or FRET signals via confocal microscopy. Thus, the amount of FRET is a direct measurement of the permanent proximity of two proteins inside a cell and is a strong indicator for direct interaction. Hum Immunol. Peripheral blood was used for this study. No, Is the Subject Area "Protein interactions" applicable to this article?
We found extremely low NK cytotoxicity in patients with low white blood cell counts using the WB assay, but relatively high NK cytotoxicity using the PBMC assay. (2017) 105:82834. Compared with the 3-h stimulation, overnight stimulation of NK cells in PBMC and WB samples led to markedly increased CD107a degranulation with all cytokine combinations (Figures 2C,E). Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis. Plasmids were isolated via the QIAamp DNA Micro Kit using the protocol for cultured cells as provided by the manufacturer or the QIAprep Spin plasmid kit (Qiagen) following the protocol Isolation of plasmid DNA from mammalian cells with QIAprep, which had better yields. Choi SI, Lee SH, Park JY, Kim KA, Lee EJ, Lee SY, et al. (2017) 78:10312. here. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases. Between-group differences were assessed using the Kruskal-Wallis test, followed by the Dunn's post analysis test to define the significance between pairs of groups. Pulmonary manifestations in Behcet disease: impaired natural killer cells activity. A positive FACS-FRET signal, however, is a strong indicator for a physiological interaction in vivo. Trotta R, Ciarlariello D, Dal Col J, Neviani P, Santhanam R, Mao H, et al. The cell surface density of CD56 and CD16 can be used to classify functionally distinct NK cell subsets: the CD56bright CD16subset (~10%), which secretes cytokines such as interferon-, activates the immune response, and the CD56dim CD16+ subset (~8090%), which directly lyses its targets (2, 3). Additionally to FRET measurements supernatants were analysed for particle production by p24 antigen ELISA and SEAP-activity 30 h post transfection. (2001) 10:45163. (c) Confocal images of 293T cells that were cotransfected with the indicated YFP (shown in green) and CFP (shown in red) fusion proteins. Kandarian F, Sunga GM, Arango-Saenz D, Rossetti M. A flow cytometry-based cytotoxicity assay for the assessment of human NK cell activity. To the contrary, coexpression of URD and CD4 resulted in a wild-type like FRET signal 75.1% (+/18.7% SD). Int J Lab Hematol. Human natural killer cells. Figure 1. Phan MT, Chun S, Kim SH, Ali AK, Lee SH, Kim S, et al. IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells. Monensin (2 M; eBiosciences) was added following a 1 h incubation, and cells were incubated for an additional 2 h. Thereafter, cells were stained with FITC-conjugated anti-human CD3 mAb and PE-Cy5-conjugated anti-human CD56 mAb for 15 min at 4C in the dark, followed by RBC lysis using 2 mL of FACS Lysing Solution. PBMCs or WB were co-incubated with K562 cells, with various cytokine combinations added to WB, for 4 h (PBMCs) or overnight (WB). Application of an improved flow cytometry-based NK cell activity assay in adult hemophagocytic lymphohistiocytosis. Using our FACS-based FRET assay we show that coexpression of CD317 and Vpu results in 39.0% (+/18.1% SD) FRET+ cells, strongly suggesting that both directly interact in living cells. Natural killer cell subsets and receptor expression in peripheral blood mononuclear cells of a healthy Korean population: reference range, influence of age and sex, and correlation between NK cell receptors and cytotoxicity. proposed a rapid, FC-based, WB cytotoxicity assay for the evaluation of NK cell function. Based on the selected optimal cytokine combinations, we developed an FC-based overnight WB NK cytotoxicity assay using cytokine activation in 28 healthy donors. This further demonstrates that our assay is sensitive to discriminate between mere co-localization in a subcellular compartment and real FRET signals. We found that NKp46 expression was significantly greater in cytokine-stimulated NK cells compared with unstimulated NK cells, whereas expression of the other receptors remained unchanged (data not shown). However, we could not reproduce their assay, and no other group has demonstrated the reproducibility of their assay (data not shown). doi: 10.1089/15258160152509073, 4. (2007) 204:2397405.
Expansion of human NK cells using K562 cells expressing OX40 ligand and short exposure to IL-21. (2014) 445:58490. (2003) 170:54649. YFP is shown in green and CFP is shown in red. Receptor cell surface expression of pEYFP-only electroporated cells was set as 100%. 293T cells were transfected with the indicated CFP/YFP fusion proteins and co-transfected with the Gag expression vector GPV-RRE (36) and a CMV-SEAP reporter construct. Yes (2019) 20:5413. doi: 10.3390/ijms20215413, 7. Lee H, Kim HS, Lee JM, Park KH, Choi AR, Yoon JH, et al. Restriction analyses of the plasmids revealed that we isolated on average 50% to 60% of false positives together with the Vpu binding partners, probably due to the presence of multiple plasmids per cell and their amplification by the SV40 large t-antigen, which is constitutively expressed in 293T cells. HIV-1 NL4-3 vpu, tat and NA7 nef as well as SIVmac 239 nef were PCR amplified from proviral DNA and ligated into pEYFP-N1 or pEYFP-C1 as C-terminal tagged fusions. CD107a expression of peripheral blood mononuclear cells (PBMCs) and whole blood (WB) under various cytokine combinations and incubation times. Hoechst B, Voigtlaender T, Ormandy L, Gamrekelashvili J, Zhao F, Wedemeyer H, et al. We were unable to compare the NKA measured by our new WB-based assay with previously used WB-based assays such as NKvue. PBMCs or WB were also incubated with cytokines overnight (1622 h) at 37C in a humidified 5% CO2 incubator, and then CD107a assays were performed as described above. (2003) 142:2926. However, this assay requires a large volume of blood and involves a PBMC isolation step. (1999) 29:165666. To address the concern that high fluorophore concentration in a specific cellular compartment (e.g. Institute of Virology, University of Ulm, Ulm, Germany, * E-mail: email@example.com. Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany, Affiliation This finding might be attributed to the effect of granulocytes, such as neutrophils, that can reduce NK cytotoxicity. 2 diabetes and prediabetes. Results: Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). Analyzed the data: CB JV DH HK MW MS. https://doi.org/10.1371/journal.pone.0009344.s002. The WB assay appeared to be more dependent on the number of NK cells in comparison to the PBMC assay. doi: 10.1126/science.1198687, 2.
(2010) A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells. Furthermore, in a setting that keeps the variety of FRET parameters constant, we could not only show that HIV-1 Vpu interacts with CD317 in living cells, but also map functional interaction domains by FACS-FRET (Fig. For electroporation, 1 g total DNA (0.3 g YFP construct and 0.7 g CFP construct) was mixed with 500,000 cells. Co-expression of CFP and YFP labelled Rev in 293T cells resulted in 66.2% (+/7.5% SD) of FRET positive cells (Fig. In contrast, deletion of the transmembrane (TM) region in Vpu (DelTM) resulted in a complete loss of FRET. Dons'koi BV, Chernyshov VP, Osypchuk VD. Most importantly, FRET measurements are generally done by fluorescence microscopy, which is tedious and essentially precludes the analysis of large cell numbers as well as high-throughput-screening (HTS) for protein interactions , . Discover a faster, simpler path to publishing in a high-quality journal. 293T or Hela cells were transfected as described above or electroporated using the Microporator MP100 device (PeqLab) as recommended by the manufacturer. Thus, this experimental can distinguish real FRET signals from cross-talk artefacts. These data suggest that the absolute NK cell count correlates more with the results of the overnight WB NK cytotoxicity assay than with those of the PBMC NK cytotoxicity assay. However, this assay is not ideal for measuring NK cytotoxicity and has not been studied in correlation with the NK cytotoxicity assay. Claus M, Watzl C. Evaluation of human natural killer cell activities in whole blood. Peripheral blood was collected in heparinized tubes from 28 healthy volunteers (15 men and 13 women, with ages ranging from 28 to 53). Kim JH, Park K, Lee SB, Kang S, Park JS, Ahn CW, et al. Yes Enumeration of peripheral blood NKp46 positive NK lymphocytes reflects NK cytotoxic activity in vitro. It has been reported that the degree of Vpu colocalization with CD317 correlates with functional repression of the restricting activity on HIV-1 release .