All antibodies were used at the manufacturers’ recommended concentrations with matched isotype controls (from Serotec). Dead/dying cells were excluded from the analysis using DAPI (Sigma) and were normally less than 5%.

Data were analysed on an LSRII flow cytometer equipped with DIVA software (BD Biosciences). The phenotypic identification selleck compound library of the ‘ex-vivo MSC’ using the CD45−/lowCD271+ phenotype was first described by our group using ICBMA [27], [28] and [35] and has since been independently validated by others [29], [36] and [37]. MSC enumeration was performed by staining the aspirated MNC fraction with CD45-FITC (Dako UK Ltd, Ely, UK), CD271-PE (Miltenyi Biotec) and 7-AAD, as previously described [28]. A minimum of 5 × 105 events were acquired and analysed using an LSRII flow cytometer to establish the percentage of CD45−/lowCD271+ cells. The frequency of CD45−/lowCD271+

per ml of sample was then calculated based on the following formula: CD45−/lowCD271+ cells/ml = % CD45−/lowCD271+ cells × MNCs/ml. Bone marrow MNCs were isolated using Lymphoprep and cells were then re-suspended at 1 × 107 cells/ml in FACs buffer. Antibodies were added at the manufacturers’ recommended concentrations and the cells were incubated for 20 min. Antibodies used were: CD45-PECy7, CD73-PE, CD34-PerCP, CD19-PE, CD33-FITC, CD61-FITC (BD Biosciences), CD90-PE, CD105-PE, CD31-FITC (Serotec) and CD271-APC (Miltenyi Biotec). The cells were washed and re-suspended http://www.selleckchem.com/products/azd4547.html in FACs buffer containing 100 ng/ml DAPI before analysing on an LSRII flow cytometer. Dead cells were excluded from the analysis using DAPI (usually < 5%) before gating on the CD45−/low CD271+ cell population and assessing the expression of all other markers. Statistical analysis

and graphing were performed using GraphPad Prism version 4 for Windows (San Diego, California, USA). Gaussian distribution could not be assumed given the number of samples and differences between donor-matched ICBMA and LBFBM groups were tested using Wilcoxon signed ranks test. The differences in the MSC content between different patient groups were analysed using Mann–Whitney test. Significance was assumed when p < 0.05. A standard CFU-F assay was triclocarban first performed to measure the MSC content of ICBM aspirates in three groups of orthopaedic patients and healthy controls (Table 1). Consistent with previously reported findings [10], high donor-to-donor variation was observed, potentially due to factors related to donor age [38] or a variable degree of dilution of ICBM sample with blood during the aspiration procedure [39]. No significant differences in CFU-F abundance in ICBMA were found between all three groups of orthopaedic patients and healthy controls (Table 1).

In prokaryotes, factors that package DNA, such as HU proteins, may control supercoiling by binding to DNA and trapping the free energy of supercoiling as writhe and subsequently releasing it through controlled dissociation [ 3 and 4]. Similarly in eukaryotes the regulated release of terminal DNA from a nucleosome, mediated by the acetylation of core histone tails, could release constrained writhe for conversion into negative supercoiling. Although in

vitro studies support this concept [ 5] its operation in vivo is elusive [ 6]. In prokaryotes and eukaryotes all activities Alpelisib cell line that require DNA to be unwound (and rewound) are potent generators of supercoiling. The classic example is the ‘twin supercoiled domain’ model where elongating RNA polymerase, in unwinding the DNA, generates positive supercoiling ahead and, in rewinding the DNA, generates negative supercoiling in its wake [7 and 8] (Figure 1). The levels of supercoiling produced in this process are prodigious, amounting to a positive and a negative

supercoil for every 10 bp transcribed. CP-868596 solubility dmso Consequently the role of topoisomerases in releasing torsional stress is crucial if the template is to be maintained in a transcriptionally competent state. Genes that are negatively supercoiled are generally more efficiently transcribed [9 and 10] but topoisomerase inhibition studies [11, 12•, 13 and 14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription. Therefore, there must be a regulated balance in the localised levels of supercoiling through the concerted actions of polymerases [15]

and topoisomerases [16 and 17]. When an activity supercoils Ribonucleotide reductase DNA the torque generated is transmitted along the molecule. If the ends of the molecule are not fixed (or at least hindered), the supercoiling will dissipate via the unhindered rotation of the helix. Therefore for supercoiling to have a structural or functional influence on DNA or chromatin it must operate within a constrained environment where the energy is at least transiently trapped or restricted. For this reason it is anticipated that genomes need to be organised into supercoiling domains with barriers that prevent the spread of topological stress. In prokaryotes the Escherichia coli genome has a hierarchical organisation based on large structural macrodomains [ 3] with the Ter domain being subdivided into smaller, 35 kb domains via MatS/MatP interactions [ 18]. This organisation establishes a dynamic structural architecture enabling packaging without interfering with transcription or replication. The genome is also separately organised into about 500 independent ∼10 kb supercoiling domains with demarcating barriers stochastically distributed and dynamically maintained [ 19 and 20].

The large size of the pooled database enabled more precise estimates of association than previous studies, particularly in stratified analyses, spline Talazoparib mw models, and assessment of interaction. Second, although pooling and harmonization of data is a substantial undertaking and requires expertise, time, and resources, individual patient data allows for many benefits over meta-analysis of published estimates, including building consistent models across studies, studying novel questions including interaction, and using novel methods of analysis such as splines. Third, the availability of 2 control groups

for comparison, that is, population-based and GERD, allows us to postulate where risk factors might be active in the pathogenesis of Barrett’s esophagus. This is important because it is feasible that a significant proportion of the population-based control group might unknowingly have Barrett’s esophagus,63 although such misclassification would bias results toward the null. Limitations of this analysis include the moderate-to-high levels of heterogeneity for some analyses. Although constituents of tobacco smoke have

changed over time,64 the studies included in this analysis Transmembrane Transporters modulator recruited incident cases and controls during a similar period (1997–2006). Regardless, constituents of tobacco smoke are likely to have differed geographically as is population susceptibility to genotoxic exposures. The unexplained Alanine-glyoxylate transaminase heterogeneity does warrant a cautious interpretation of summary estimates, although associations were largely consistent in a majority of studies included, and similar summary estimates with low heterogeneity were estimated when the study that was the source of the most heterogeneity was omitted from analysis. Another limitation is the possibility of recall bias, given the case-control design of the included studies, although the intensity and duration of smoking are usually recalled relatively reliably.65 Lastly, we did not adjust for dietary

variables in this analysis; although previous studies suggest that diet has minimal effects on relationships between smoking and Barrett’s esophagus, there remains the possibility of residual confounding through diet and other exposures. In conclusion, cigarette smoking is a risk factor for Barrett’s esophagus, with adjusted ORs for multiple measures of association in the 1.5 to 2 range. The association appears to strengthen with increased exposure to cigarette smoking until approximately 20 pack-years, where it begins to plateau. If smoking is a causative agent of Barrett’s esophagus, it is an attractive modifiable risk factor, especially in high-risk groups such as elderly, obese males with GERD symptoms.

The third spawning bed, which is dominated by F. lumbricalis, was visited twice within a period of three days at the end of April 2009. The sample taken on 21 April 2009 contained eggs AZD8055 mouse in the embryonic developmental stages (m–n), and 3 days later the eggs had embryos already in developmental stages (o–p). According to Rajasilta et al. (1989, 1993, 2006) red algae (including F. lumbricalis) have a negative effect on Baltic herring eggs, causing higher egg mortality. However, in this study, the embryos in the eggs collected from F. lumbricalis thalli developed normally to the very last developmental stages, resulting in successful mass hatching. One of the advantages of F. lumbricalis as a spawning

substrate could buy Epacadostat be the extensive 3D structure of the firm F. lumbricalis thalli. This can accommodate a larger number of eggs while ensuring their proper aeration compared with other spawning surfaces, on which eggs may be laid in multilayers. It is known that embryonic oxygen uptake increases in the later development stages ( Silva & Tytlerb 1973); in multilayer mats only the eggs in the upper layers develop successfully to the last stages, whereas the eggs in the deeper layers abort (the abortion stage is layer-dependent: the deeper the egg, the earlier the abortion stage) and/or show severe embryonic abnormalities ( Messieh & Rosenthal 1989), most likely due to the lack of

oxygen. This is less likely to happen when F. lumbricalis is used as a spawning substrate. The locations of Baltic herring spawning beds are

usually very specific (Geffen 2009), and there are reports that Baltic herrings return to the same spawning beds year after year (Oulasvirta & Lehtonen 1988, Bergstrm et al., 2007). This occurs even if there is a strong anthropogenic impact in the area (Rajasilta et al. 2006). During this study the hydrological conditions between two spawning seasons were very different: in 2009 there was strong upwelling resulting in colder water and higher salinity. In 2010 the upwelling was not significant and the water in the coastal area was mixed with hyper-eutrophic Curonian Lagoon waters, resulting in greater turbidity and lower salinity. Moreover, the winter in 2010 was much colder and longer compared with 2009, resulting in later spawning (Figure 4). Despite these differences, 4��8C the spatial pattern of the spawning persisted, indicating that there are more stable factors determining the distribution of the spawning beds than just the hydrological conditions. One such factor could be the bottom geomorphology, which was tested in this study in terms of the bottom slope. Table 3 shows the average 100 m profile slopes to the east and west of the sampling points, the average profile depth gradients as well as the average maximum westward and eastward slopes values for 10 m segments with corresponding standard deviations. Graphical representations of the bottom profiles are shown in Figure 5.

The notion that new encoding and prior knowledge interact with one another is by no means new 6 and 7; yet, the neural mechanisms and behavioral implications of memory integration have only recently become the subject of empirical investigation. The field’s growing interest in understanding these complex, real-world aspects of episodic memory has been realized thanks to the

introduction of elegant behavioral paradigms and advanced analysis methods for neural learn more data (see example in Figure 1b). We first review evidence for the neural mechanisms that support memory integration. We then turn to a discussion of the range of behaviors that might be supported by integration, from flexible navigation to imagination and creativity. Finally, we set forth questions for future research. Human and animal lesion work highlights the critical roles of the hippocampus www.selleckchem.com/products/pirfenidone.html [8] and medial prefrontal cortex (mPFC 9 and 10) in memory integration (Figure 2). Damage to these structures impairs the ability to combine information acquired during different episodes despite intact memory for previously

learned events. However, while these data underscore the importance of hippocampus and mPFC in memory integration, the precise mechanisms by which these regions contribute have only recently started to become clear. One period during which memory integration may take place is when new learning experiences share content (e.g., a person, place, or thing) with existing memory

traces (Figure 1a). For a discussion of specific factors that impact the likelihood of integration, see Box 1. During the new experience, pattern completion mechanisms supported by the hippocampus reactivate the previously stored, overlapping memory 11 and 12. Empirical support for reactivation of prior memories during overlapping learning experiences ZD1839 ic50 has recently been garnered using neural decoding of fMRI data (Figure 1b) 4••, 5 and 13. A number of studies have investigated the various factors that influence integration. For instance, while there is evidence that integration can occur in the absence of conscious awareness 34, 38••, 52 and 53, studies have shown that integration may be facilitated when subjects become aware of the task structure (either via instructional manipulations or spontaneously) [54]. In fact, one experiment [54] demonstrated that such knowledge specifically benefitted judgments that spanned episodes with no effect on memory for the individual episodes themselves, suggesting that integration does not necessarily emerge with effective encoding of the underlying experiences. One possibility is that awareness constrains mental models in prefrontal regions, which in turn biases hippocampal reactivation during learning toward task-relevant memories, allowing for integration across events.

The images acquired with Cellomics™ Arrayscan® were analyzed by Spot Detector

V4 BioApplication. Neutral lipid accumulation: Cells were washed twice with HBSS (+Ca2+/Mg2+), stained with Hoechst 33,342 www.selleckchem.com/products/Metformin-hydrochloride(Glucophage).html and BODIPY® 493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) (2 μM in DMSO) (Invitrogen, USA) and incubated 15 min at 37 °C. The images acquired with Cellomics™ Arrayscan® were analyzed by Compartmental Analysis V4 BioApplication. Phospholipids accumulation: Cells were washed twice with HBSS (+Ca2+/Mg2+) and stained HCS LipidTox™ Red (1:1000 in culture medium) (Invitrogen, USA) for 24 h at 37 °C in culture medium. After 24 h, the cells were washed 3 times with HBSS (+Ca2+/Mg2+), stained with Hoechst 33,342 and incubated 10 min at 37 °C. The images acquired with Cellomics™ Arrayscan® were analyzed by Spot Detector V4 BioApplication. FastLane Cell Multiplex Kit (200), (Qiagen, USA) was used to isolate first-strand cDNA directly from cultured cells without RNA purification according to manufacturer’s instructions. RT–PCR was performed using a StepOnePlus™ Instrument (Applied Biosystems, USA) selleck screening library in the presence of TaqMan® Gene E probes (Table 1) (Applied Biosystems, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. A volume of

20 μl was the used for each reaction. Relative gene expression was analyzed using the 2−ΔΔCt method. Statistical comparisons were performed between each dose group and the control using two-way ANOVA. Values were first normalized within each experiment in percent of control, to make the experiments comparable. The data were obtained from 3 independent experiments, each of them consisting

of 3 replicates. Statistical analysis was conducted using Graph Pad Prism 6 software. Differences compared to respective daily controls were considered as statistically Nintedanib (BIBF 1120) significant for *p < 0.05. Following isolation, primary rat hepatocytes were purified and cultured in Collagen I-coated plates. After the addition of a layer of Matrigel™, hepatocytes showed typical cuboidal morphology within the same day (Fig. 1A), whereas the canalicular networks were visible only after 2–3 days in culture (Fig. 1B). After 8 days in culture rat hepatocytes started to lose their cuboidal morphology and acquired spindle-like shape (Fig. 1C and D) together with dead cell detachment from the wells (Fig. 1E). In contrast, cells receiving a second layer of Matrigel™ on day 7 showed significant improvement of the culture quality. The cells maintained their morphology together with a lower disruption of the canalicular networks after 8 days (Fig. 1H–J).

The reverse primer was 30Rec antisense (5′-CGGGATCCTTATTTCTTGAATGTCACCCA-3′), which contains a BamH I restriction site (underlined) and a stop codon (bold). The obtained PCR product was cloned into the pGEM-T vector (Promega, Madison). The pGEM-T vector containing the cDNA encoding the mature protein was then digested with the Xho I and BamH I restriction enzymes. The excised insert was gel purified using the QIAquick Gel 74 Extraction Kit (Qiagen, Valencia) and

subcloned into a pET-14b vector (Novagen, Madison) digested with the same enzymes. The recombinant protein GFP-LiRecDT1 was obtained by subcloning the previously constructed LiRecDT1 sequence and the enhanced green fluorescence protein (EGFP) sequence into pET-14b using a Blunt-Cut-Cut strategy at the Nde I site of pET-14b and two BamH I sites (between LiRecDT1, EGFP

and the vector) ( Chaves-Moreira et al., 2009). All recombinant constructs NVP-BKM120 were expressed as fusion proteins with a 6x His-Tag at the N terminus and a 13 amino acid linker (including a thrombin selleckchem site) between the 6x His-Tag and mature protein (N-terminal amino acid sequence before the mature protein: MGSSHHHHHHSSGLVPRGSHMLE). pET-14b/L. intermedia cDNA constructs were transformed into One Shot E. coli BL21(DE3)pLysS competent cells (Invitrogen, Carlsbad) and plated on LB agar plates containing 100 mg/mL ampicillin and 34 mg/mL chloramphenicol. A single colony was inoculated into 50 mL of LB broth (100 mg/mL ampicillin and 34 mg/mL chloramphenicol) and grown overnight at 37 °C. A 10 mL aliquot of this overnight culture was grown in 1 L of LB broth/ampicillin/chloramphenicol at 37 °C until an OD of 0.5 at 550 nm was reached. IPTG (isopropyl b-d-thiogalactoside) was added to a final concentration of 0.05 mM, and the culture was induced by incubation for an Cediranib (AZD2171) additional 3.5 h at 30 °C (with vigorous shaking). Cells were harvested via centrifugation (4000 g, 7 min), and the pellet was frozen at −20 °C overnight. Cell suspensions were thawed and then disrupted via 6 cycles of 10 s of sonication at low intensity. The lysed materials were centrifuged (20,000 × g, 20 min), and the supernatants were incubated with 1 mL of Ni2+-NTA agarose

beads for 1 h at 4 °C (with gentle agitation). The suspensions were loaded into a column, and the packed gel was thoroughly washed with the appropriate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl, 20 mM imidazole) until the OD at 280 nm reached 0.01. The recombinant protein was eluted with 10 mL of elution buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl, 250 mM imidazole), and 1 mL fractions were collected and analyzed via 12.5% SDS-PAGE under reducing conditions. The fractions were pooled and dialyzed against phosphate-buffered saline (PBS). Protein concentrations were determined using the Coomassie Blue method. Five replicates were performed. Protein analysis was conducted using an IEF system (Ettan IPGphor 3, GE Healthcare) for the first dimension and 12.

, China). After electrophoresis, the DNA fragments were transferred to a nylon membrane (Amersham Biosciences Shanghai Ltd., Darmstadt, Germany). Pre-hybridization was performed at 42 °C 2 h. The probe was denatured at 100 °C check details for 10 min, then quickly cooled in an ice bath for 5 min, and 4.0 μL of denatured probe in 8.0 mL

hybridization solution (Hyb-100) was added. The hybridization step was performed in a hybridization oven at 42 °C overnight. The washing and detection steps were performed according to the kit instructions. Three biological replicates were conducted, and two technical replicates were analyzed for each biological replicate. The oligonucleotide primers and TaqMan fluorescent dye-labeled probes were designed in ABI Prism Primer Express Version 3.0 software (Applied Biosystems, Foster City, USA). All primers and fluorogenic probes were synthesized by Shanghai Sangon Co. Ltd. (Shanghai, China). The plant universal primer cob-F/R was used to evaluate the DNA quality. The primer Lhcb2-1F/1R was used for qualitative and quantitative PCR to detect the Lhcb2 gene with the probe Lhcb2-P; Lhcb2-2F/2R was used for Southern blot probe labeling. The nucleotide sequences and product sizes of the primers are listed in Table 1. For qualitative detection, PCR was carried out Entinostat clinical trial in final volumes

of 30 μL containing 1× reaction buffer (50 mM KCl, 10 mM Tris–HCl, pH 8.3, and 1.5 mM MgCl2), 0.2 mM dNTPs, 0.3 μM of each primer, 2.5 units of Taq DNA polymerase (TaKaRa Biotechnology Co. Ltd., China), and 1 μL DNA template. All amplifications were carried out

on an ABI2720 thermal cycler (Applied Biosystems, U.S.A.) as follows: one step of 5 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C, and one step of 5 min at 72 °C. For cob gene amplification, a template concentration of 100 ng/μL was used; for the species-specific gene amplification, the template was 10-fold serially diluted from 100 ng/μL to 1 pg/μL. The products were analyzed by 2% agarose gel electrophoresis (1× TAE) and stained with ethidium bromide. Three biological replicates were conducted, and three technical replicates were analyzed for each biological replicate. Real-time PCR reactions were performed using an ABI7500 Real-Time PCR System instrument (Applied Biosystems, U.S.A). Amplification Adenylyl cyclase specificity was evaluated in reaction volumes of 25 μL containing 1× RealMasterMix SYBR Green (TIANGEN, China), 100 nM primers, and 50 ng DNA with the following program: 2 min at 50, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C, followed by melting curve analysis. The temperature program used for the melting curve analysis was 60–95 °C with a heating rate of 0.5 °C per second and a continuous fluorescence measurement. Each sample was quantified in duplicate for each biological replicate, and three biological replicates were conducted.

This down-regulation allows Lrp5 to be instead bound by Wnts, which may already be present or may have been up-regulated by the mechanical loading [107], and the result is activation of the Wnt/β-catenin signaling pathway. The reports at the beginning of the last decade demonstrating that mutations in LRP5 are causally associated with changes in human bone mass stimulated extensive research into understanding the underlying mechanisms. This work demonstrated that components of this pathway, including LRP5, are required for osteocytes to Fluorouracil ic50 respond to mechanical load. In addition, regulation of secretion of the Wnt inhibitor, SOST, from osteocytes

plays a key role in coordinating the response to these mechanical signals. However, there are several outstanding questions remaining to be addressed. For example, what is the mechanism by which LRP5 is activated via mechanical loading? Does this involve a Wnt ligand? If so, which one(s)?

Answers to these questions will further inform the development of therapies based on activating this pathway to treat osteoporosis and other bone diseases. buy JQ1 The authors thank David Nadziejka for technical editing of the manuscript and Michaela Kneissel for comments. Work in the Williams Laboratory is supported by National Institutes of Health grant AR053293 (BOW) and by Van Andel Research Institute. The authors declare that they have no conflicts of interest. “
“Osteocytes represent the terminally differentiated state of the osteoblast lineage and are embedded within the mineralized bone matrix. Because they are trapped within a mineralized “prison”, osteocytes are not easily accessible and therefore our understanding of their role in bone remodeling remains incomplete. Advanced imaging techniques (ex vivo and in vivo) and

the exploitation of in vivo models to extract Thiamet G quantitative biochemical information are tools which are beginning to provide more clues about both the anatomy and biology of osteocytes, respectively. Synthesis of these data will therefore greatly facilitate a more complete understanding of the osteocyte’s function. Ex vivo imaging of osteocytes has proved challenging due to the need to develop methodologies for imaging and sectioning of undecalcified specimens or to develop protocols for decalcifying specimens to enable conventional sectioning and imaging techniques to be used. Early imaging approaches relied mainly on staining of the lacuno-canalicular network (LCN) rather than the osteocyte itself using histological stains combined with conventional light microscopy. With the advent of confocal imaging approaches it has become relatively straightforward to image osteocytes and their lacuno-canalicular system three-dimensionally (3D) in situ within their bone environment.

Therefore, there is a large unmet medical need to develop a simple and accurate assay that can overcome these limitations and provide clinicians with valuable quantitative measurements that they can then use to optimize the management of patients on biologic therapies. Here, we have developed and validated a novel homogenous mobility shift assay (HMSA) using

size-exclusion high-performance liquid chromatography (SE-HPLC) to quantitatively measure both induced antibodies-to-infliximab (ATI) levels and IFX levels in serum samples collected from IBD patients being treated with IFX. Individual serum samples from healthy controls were obtained from Y-27632 in vitro blood bank donors (Golden West Biologics, Temecula, CA). Sera from IBD patients treated with IFX were obtained from residual samples leftover after testing for ATI and IFX levels in our laboratories and the patient information was de-identified. Unless

otherwise noted, all reagents and chemicals were obtained from either Thermo Fisher Scientific (Waltham, BMS-354825 in vitro MA) or Sigma Aldrich Corporation (St. Louis, MO). Commercially-available infliximab (RemicadeTM, Janssen Biotech, Inc., Horsham, PA) was buffer exchanged with phosphate buffered saline (PBS, pH 7.3) and labeled with AlexaFluor 488 (Life Technology, Carlsbad, CA) following the manufacturer’s instructions. Briefly, a reaction mixture consisting of 10 mg of IFX, 154 μg of AlexaFluor 488 dye, and 1 mL 1 × PBS (pH 8.0) was incubated in the dark at room temperature (RT) for 1 h with constant stirring. A desalting column was then used to remove free AlexaFluor 488, and the infliximab-AlexaFluor 3-oxoacyl-(acyl-carrier-protein) reductase 488 conjugate (IFX-488) was collected. The protein concentration and labeling efficiency of the conjugate was measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The NanoDrop spectrophotometer measures the A280 value for the protein concentration and the A494 value for AlexaFluor 488 concentration. The approximate molar extinction coefficient of the AlexFluor 488 dye at 494 nm is 71,000 cm− 1 M− 1 and the labeling efficiency

is calculated as follows: molesdyepermoleprotein=A494×dilutionfactor71,000×proteinconcentrationM Only those conjugates containing 2 to 3 fluorescent dyes per antibody qualified for the ATI-HMSA. The procedure for the labeling of recombinant TNF-α (RayBiotech, Inc, Norcross, GA) with AlexaFluor 488 was identical to that used for the labeling of IFX. The molar ratio of TNF-α to fluorescent dye in the reaction mixture was 1:6 and the resulting TNF-α-AlexaFluor 488 conjugate (TNF-488) contained 1–2 dye molecules per TNF-α. Activated AlexaFluor 488 (1 mg) and 4 mL 1 M Tris buffer (pH 8.0) were mixed for 1 h on a magnetic stirrer at RT to block the active site on the dye. The resulting solution was buffer-exchanged with 1 × PBS. The blocked AlexaFluor 488 was used as the IC and combined with either IFX-488 or TNF-488 at a molar ratio of 1:1.