9%. Two hours later, mice were sacrificed by cervical dislocation and back skin was totally removed in order to measure the area of the hemorrhagic lesion. MHD was defined by the dose causing a lesion with a diameter of 10 mm. PLA2 activity was measured using an indirect hemolytic assay (Gutierrez et al., 1988). Increasing concentrations of B. andianus venom (from 0.004 μg up to 10 μg) were prepared in a final volume of 15 μl in PBS and added to 2 mm wells in agarose gel plates (0.8% in PBS, pH = 8.1, containing 1.2% sheep erythrocytes, 1.2% egg yolk and 100 mM CaCl2). Plates were incubated at 37 °C for 18 h and the diameters of the hemolytic haloes were measured.

In controls, 15 μl of PABA was used. One unit (Minimum PLA2 Doses-MPD) corresponds to a minor concentration isocitrate dehydrogenase signaling pathway of venom which produced a hemolytic halo of 10 mm diameter. Experiments

were conducted in triplicate. Proteolytic p38 MAPK cancer activity was measured with dimethylcasein (Sigma) as described in Lin et al. (1969) with the modifications described in Sanchez et al. (2000). Dilutions corresponding to 5, 10, 20 and 40 μg of venom were used and absorbance values were determined at 340 nm. One unit was defined as ΔA 340 nm/min. Activity was expressed relative to protein concentration (mg). The anti-venom potency was determined by mixing 5LD50 of B. andianus venom with 12.5, 25, 50, 100 or 200 μl of PABA and incubating for 1 h at 37 °C followed by i.p. injection in 5 groups of 4 mice. Median effective dose (ED50) was calculated from the number of deaths within 24 h of injection of the venom/anti-venom mixture using Probit analysis as described above. The ED50 was expressed as ml anti-venom/mg of venom needed to prevent death in 50% of the injected mice. To determine the neutralization of hemorrhagic activity, PABA was incubated with either Sorafenib in vitro 3MHD or 5MHD for 30 min at 37 °C according to manufacturer’s

instructions (1 μL of serum to 2.5 μg of venom) and inoculated in different groups of 3 Swiss male mice (18–22 g) as described above. Positive and negative control groups, each consisting of 2 mice were treated with venom alone (5MHD) or anti-venom, respectively. Two hours later, mice were euthanized and the hemorrhage was measured (Kondo et al., 1960; Sanchez et al., 1992). Inhibition of PLA2 activity of B. andianus venom by PABA was conducted as described by Gutierrez et al. (1998). Two MPD of venom were incubated with 13, 6.5 and 3.25 μl of anti-venom for 30 min at 37 °C, and 15 μl of each mixture added in triplicate to wells in agarose gels. Neutralization of venom was checked by the absence of halos on the plate’s surface. Inhibition of dimethylcasein hydrolysis by PABA was estimated by incubation (30 min at 37 °C) of a fixed concentration of B. andianus venom with increasing amounts of anti-venom (μl). After incubation the mixtures were tested as described before.

All spectra were obtained in the positive-ion mode. STA-9090 concentration Data acquisition and deconvolution of data were performed on Xcalibur Windows NT PC data acquisition system. OcyKTx2 was compared against all α-KTxs described until now (for a complete list see http://www.uniprot.org/docs/scorpktx). Multiple sequence alignments were performed by ClustalW XXL (at http://embnet.vital-it.ch/software/ClustalW-XXL.html) followed by manual adjustment. This result was subsequently used to build phylogenetic analysis and consensus sequences. In the sequence matrix, all positions containing gaps and missing data were eliminated. The Maximum

Parsimony method with 500 Bootstrap replications and Close–Neighbor–Interchange algorithm model on MEGA 5 software were used in the reconstruction of the phylogenetic tree. The analysis involved 124 amino acid sequences. Insect Sf9 cells were grown at 27 °C in Grace’s

media (Gibco BRL). The cells were infected with a multiplicity of infection of 10, with a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus) containing the cDNA of Shaker-B K+-channels. Electrophysiological recordings were conducted 48–72 h after the infection, as previously reported [26]. Macroscopic currents were recorded with the whole cell configuration of the patch-clamp technique, with an Axopatch 1D (Axon Instruments, Inc.). The currents were filtered Navitoclax research buy at 5 kHz and sampled every 100 μs with a DigiData 1200 interface (Axon Instruments, Inc.). Electrodes were pulled from borosilicate glass (KIMAX 51) to

a 1–1.5 MΩ resistance. 80% of the series resistance was electronically compensated. The holding potential used throughout the work was −90 mV. The recording solutions were: external bath (in mM): 145 NaCl, 10 Ca2Cl, buffered with 10 HEPES-Na at pH 7.2; internal pipette solution (in mM): 90 KF, 30 KCl, 10 EGTA, buffered with 10 HEPES-K at pH 7.2. Lymphocyte separation: Kv1.3 currents were measured in human peripheral T lymphocytes. Heparinized human peripheral venous blood was obtained from healthy volunteers. Mononuclear cells were separated by Cell Penetrating Peptide Ficoll–Hypaque density gradient centrifugation. Collected cells were washed twice with Ca2+- and Mg2+-free Hanks’ solution containing 25 mM HEPES buffer, pH 7.4. Cells were cultured in a 5% CO2 incubator at 37 °C in 24-well culture plates in RPMI 1640 medium supplemented with 10% fetal calf serum (Sigma–Aldrich Kft, Budapest, Hungary), 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine at 0.5 × 106/mL density for 3 to 4 days. The culture medium also contained 2.5 or 5 μg/mL phytohemagglutinin A (Sigma–Aldrich Kft, Budapest, Hungary) to increase K+-channel expression [11]. For the measurement of ionic currents standard whole-cell patch-clamp procedures were performed. The bath solution consisted of (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 5.5 glucose, and 10 HEPES, pH 7.35, supplemented with 0.

Interestingly, rhLK8 as a single agent significantly reduced tumor size, and this effect was more pronounced

in HeyA8 tumors producing low levels of VEGF. rhLK8 appears not to target tumor cells but inhibits activated endothelial cells. Therefore, antitumor efficacy of rhLK8 was independent on the VEGF expression of the corresponding tumor cells. Because SKOV3ip1 produced the profound amount Alpelisib supplier of biologically active VEGF, they have more biologic redundancy in the survival of tumor-associated endothelial cells than HeyA8, which depends on more tight and narrower angiogenesis activity. Therefore, when rhLK8 inhibits angiogenesis of tumor-associated vasculature, there would be more impact on the HeyA8 tumors. This may explain the synergistic therapeutic effect of rhLK8 on HeyA8 cells. Collectively, these results suggest that VEGF may not be a determinant of the response of certain cancers to antiangiogenic therapy with rhLK8. In this study, expression of VEGF was increased in HeyA8 tumors of mice treated with paclitaxel or rhLK8. This may be from

the local hypoxic condition induced by impaired tumor vasculature caused by either destruction of proliferating tumor-associated endothelial cells by paclitaxel or suppressed angiogenesis by rhLK8. Because intrinsic level of expression in SKOV3ip1 tumors was high, it was not altered by the local hypoxia induced

by the treatment with either paclitaxel or rhLK8. Decreased expression of VEGF in tumors of mice treated by the selleck chemical combination of paclitaxel and rhLK8 may reflect the decreased biologic activity or, further, viability of tumor cells from additive or synergistic effects on the induction of the apoptosis of tumor-associated endothelial cells. Previously, we showed that combination treatment with paclitaxel and rhLK8 Neratinib chemical structure could be an effective therapy for prostate cancer metastatic to the bone [19], as well as other solid tumors including colorectal carcinoma, pancreatic carcinoma, renal cell carcinoma, and melanoma (data not shown); however, macroscopic (tumor incidence and tumor size) or microscopic (cellular proliferation, MVD, or apoptosis) responses to therapy with paclitaxel and rhLK8 were not observed in orthotopic animal models of human lung cancer, PC14 cells, which produce high levels of VEGF, and pleural effusion (Kim JS et al., unpublished data). The mechanisms mediating the different responses to rhLK8 treatment between tumors growing in different organs are not clear. One possible explanation is that rhLK8 may have a differential effect on the biologic properties of tumor-associated endothelial cells, because the specific features of endothelial cells have been reported to be organ-dependent [39] and [40].

Based on several lines of evidence, Estes et al. (1998) concluded that killer whale (Orcinus orca) predation was the most parsimonious explanation for this decline. Garshelis and Johnson (1999) commented that equally compelling evidence BIBW2992 research buy for killer whale predation on otters existed for Knight Island. Common to both Knight Island and the Aleutians, there was no indication of reduced birthing or pup survival, few dead otters washed ashore (as they would in cases of disease, malnutrition, winter mortality,

or contamination), and body condition of otters indicated that food supplies were adequate ( Dean et al., 2000, Dean et al., 2002 and Laidre et al., 2006). In the Aleutians, only six killer whale attacks have been observed, and among these only three of the otters died (Hatfield et al., 1998). However, given the low probability of actually witnessing such brief events in this huge area, the three confirmed mortalities were extrapolated to an estimated 40,000 otters consumed by killer INCB024360 clinical trial whales (Estes et al., 1998). It is now widely believed that killer whale predation reduced the Aleutian Islands’ otter population by more than 95% (Estes et al., 2005). Doroff et al. (2003, p. 55) called it “one of the most widespread and precipitous population declines for a mammalian carnivore in recorded history.”

Despite the rather scant observational evidence of the cause for this decline, when southwestern Alaska sea otters were proposed as a threatened population under the U.S. Endangered Species Act, killer whale predation was considered the most probable cause, with little support for other alternatives (U.S. Federal Register, 2004). Although intensive studies of both otters and killer whales have been conducted in PWS since

the early 1980s, the first attack was not witnessed until 1992 – by coincidence, shortly after the spill. All three observed killer whale attacks since then occurred at Knight Island, two of which were in Herring Bay, NKI (Hatfield et al., 1998). Additionally, in 2003, a killer whale was found dead in LaTouche Passage, south of Knight Island, with five sea otters in its stomach. This whale was identified as part of a pod whose range was centered in the Knight Island area (Vos et al., 2006). Killer whales could not only consume Protein kinase N1 several otters per day at Knight Island, but the risk of predation could drive otters to move to safer areas. Many scientists have moved beyond the question of whether killer whales began preying heavily on sea otters to why they did. Leading theories suggest that those killer whales that preferentially prey on marine mammals (rather than fish) have been forced to switch from diminishing stocks of harbor seals (Phoca vitulina) and Steller sea lions (Eumetopias jubatus) to much smaller, less-preferred sea otters. Although harbor seals are a preferred prey of most marine-mammal eating killer whales, in PWS whales prey equally on Dall’s porpoise (Phocoenoides dalli).

With regard to the other correlational analyses, we found significant (two-tailed) relationships between experienced anxiety and psychological hardiness (total, commitment, and control). One aim of this study was to determine whether characteristics of psychological hardiness mediated the relationship between traits of psychopathy and experienced anxiety in a prison setting. Like the

correlation analyses, our mediation analysis (see Table 2 and Fig. 1), ALK inhibitor did not reveal any significant direct relationship between either F1 or F2 and anxiety. We did, however, find significant indirect effects mediated through the commitment dimension for both F1 and F2, but in reverse directions. This finding points to characteristics of commitment as a partial mediator of the relationship between psychopathy and anxiety. The opposite direction effects for F1 and F2 emphasize the heterogeneity of the psychopathy construct. Partly through high levels of commitment, F1 traits (interpersonal and emotional detachment) seem to protect against anxiety, while F2 traits (unstable and antisocial), partly through lower levels of commitment, seem to be a risk factor for experiencing anxiety. While interesting, it is important to note that the mediation effect of commitment is Cabozantinib manufacturer only partial, with a modest effect

size (F1 k2 = .112; F2 k2 = .155). However, by explaining a little over one-tenth of the relationship, it still represents a significant contribution that has not previously been shown. Our findings concerning how personality variables (i.e., psychopathy and psychological hardiness) are associated with experienced anxiety in a prison setting might suggest that the stressor of incarceration does not affect the psychological well-being of all individuals equally (Bukstel & Kilmann, 1980). Traits of both psychopathy and

psychological hardiness seem to act as resiliency factors in relation to anxiety that might also act as a buffer against other adverse health effects of stress. This protective feature only seems to be related to some characteristics of psychopathy, however, namely interpersonal and emotional ID-8 detachment (PCL-R F1). This resiliency against anxiety related to F1 seems to correspond to Cleckley’s original connotation of psychopathy, and to what is also called primary psychopathy (Cleckley, 1976, Karpman, 1948 and Skeem et al., 2011). That PCL-2 F2, with its focus on antisocial behavior, is found to be more positively related to anxiety coincides with other findings of strong comorbidity between Antisocial Personality Disorder (ASPD) and anxiety disorders (Goodwin & Hamilton, 2003). Antisocial behavior can also be a symptom/indication of other mental disorders, including anxiety (Goodwin and Hamilton, 2003 and Karpman, 1948).

Van Buren and Fedio (1976) applied DES in 60 Hz pulses with a total duration of 2.5 msec, with a current of 1 mA. Lüders et al. (1987) applied pulses of .3 msec duration in 50 Hz trains of 5–10 sec. For each electrode, the applied current was increased in .5 or 1 mA steps. Stimulation was stopped when i) a response was obtained, ii) after discharges were observed or iii) the arbitrary limit of 15 mA was reached. Most subsequent studies used 5-FU mouse similar stimulation parameters, with

the exceptions of Fried et al. (1991), who applied .1 msec pulses; and Chauvel et al. (1996), who applied pulses of 1 msec duration. The final stimulation current is rarely reported. NMAs will only be found if the electrode of interest

is stimulated during an ongoing action of the appropriate musculature. Moreover, NMAs were not the main interest of many of these studies. In some cases, they are reported anecdotally, as incidental findings. Accordingly, the probability of finding an NMA depends on how many alternative movements Dasatinib price the experimenter tries to arrest. Since many of the reported NMAs involve inhibition of a single type of motor response, it seems likely that many possible NMAs may be missed, due to sparse sampling (see Effector specificity, below). Nevertheless, NMAs are surprisingly common, and 3% (Chassagnon et al., 2008) to 35% (Nii et al., 1996) of stimulation sites have been classified as NMAs. A typical procedure involves asking the patient to read a text out loud and then serially stimulating all electrodes (Lüders et al., 1988, Lüders et al., 1992 and Penfield and Jasper, 1954). If and only if speech arrest effects are found, inhibition of other motor actions from the same site is then evaluated. Unsurprisingly therefore, speech arrest is the most frequently reported negative

motor response, while NMAs for non-speech movement are relatively rare. This may represent an artefact of the sampling procedure, Selleckchem Forskolin rather than a fundamental feature of neural organisation of action inhibition. The screening protocol based on reading aloud also overemphasises the overlap between speech and non-speech NMAs, and thus underestimates any actual effector specificity of NMAs. Stimulation at a given cortical site generally produces negative motor responses in a restricted set of muscles only, without affecting the ability to make other voluntary movements (Chassagnon et al., 2008 and Hanakawa et al., 2001; Ikeda et al., 1999, Lim et al., 1994, Mikuni et al., 2006 and Penfield and Rasmussen, 1950). That is, NMAs can sometimes be effector-specific. Negative motor effects are predominantly contralateral. Further, negative motor responses were in some cases stronger and more frequent for distal muscles than for proximal ones, and for fingers as opposed to toes (Lüders et al., 1992). This suggests an effector-specific organisation of motor inhibition.

There was no evidence for titer–age interactions. The number of participants with ILI confirmed as influenza was small (Fig. S1), and associations between DNA Damage inhibitor HI titer and illness amongst those infected were not significant, although there was trend for participants who developed ILI after H3N2 infection in season 2 to have lower pre-season titers (Fig. S3). To further investigate whether non-HI antibodies contribute to protection against infection we assessed the effect of infection in S1 or S2 on infection in S2 or S3 respectively, when the first infection did not induce HI antibodies to the second infection (Table 3). This analysis was limited to comparisons across different subtypes

with the exception of H1N1 in S2, which was not associated with production of HI antibodies to pandemic H1N1 in S3 (p = 0.921). Associations between influenza A and B infections were investigated to verify whether effects reflect adaptive antibody responses as opposed to non-specific mechanisms. For S2, there was no detectable effect

of prior H1N1 infection on subsequent H3N2 infection or vice versa but the numbers infected were small and confidence intervals were large, particularly for the effects of Cetuximab datasheet H3N2. However, infection with H1N1 in S2 was associated with a clear reduction in the risk of pandemic H1N1 infection in S3, whereas B (Yamagata) had the opposite effect and H3N2 had no significant effect. There was no similar effect of B in S1 on H1N1 in S2 despite similar sample sizes. The effects of H1N1 and B infection in S2 on pandemic H1N1 infection in S3 were maintained after adjusting for age and pre-season HI titer, and when both prior H1N1 and B were included together in the same model. In subjects whose influenza immunity has been shaped by prior natural infection without vaccination, protection

against infection was significantly associated with homologous HI titer for H3N2 and B Yamagata lineage but not for H1N1. However, protection against H1N1 infection was associated with increasing Histone demethylase age, and protection against pandemic H1N1 was also associated with prior confirmed seasonal H1N1 infection, even though HI antibodies were rarely detected. It was also clear that HI antibodies were not always induced following H1N1 infection and titers induced were low relative to H3N2 infection. The lower levels of H1N1 HI seroconversion following virologically confirmed infection means that we may have underestimated the proportion of participants that were H1N1 infected, and this potential under-ascertainment of infections would be concentrated amongst those with low baseline titers. This could be one factor that decreases the likelihood of detecting a significant protective effect of H1N1 HI titers, but also indicates a difference between the subtypes with respect to HI antibody.

, 2007), but to our knowledge no similar network has been identified in the left hemisphere. A recent meta-analysis suggests that right pre-SMA is more strongly activated in response to increased task selleck kinase inhibitor difficulty – situations which are very likely to involve an element of selection or response switching (Keuken et al., 2014). Therefore it appears that there is evidence to suggest that

left and right pre-SMA may perform different functions, but how much these reflect hemispheric specialisations and differences in task design remains an open question. This discussion has focused on the role of pre-SMA and SMA in stopping and switching response plans. Other regions within medial frontal cortex, particularly ACC, have also been implicated in stopping responses (Botvinick et al., 1999). Lesion studies have demonstrated functional heterogeneity within ACC, with the behavioural deficits dependent on the modality of response (Turken & Swick, 1999), and more often associated check details with deficits in error detection and correction (Ullsperger & von Cramon, 2006). The Eriksen Flanker differs fundamentally from the STOP and CHANGE paradigms because it activates conflicting responses simultaneously, analogous to the Stroop effect, rather than via two separate stimuli presented at different temporal intervals. This may explain why we did not observe any significant behavioural deficits on this paradigm, except generalised slowing. These data

might arguably be considered to be consistent with the proposal that ACC does not activate when only stimulus selection is required, but instead appears to provide an evaluative and error monitoring function in situations of conflict (Rushworth et al., 2004 and Swick and Turken, 2002). In conclusion, our finding of a dissociation between stopping and switching actions following a lesion of caudal pre-SMA sheds new light on the role of this brain area in the control of action. The results suggest that caudal pre-SMA plays an important role in facilitating selective inhibition, either by promoting this L-gulonolactone oxidase directly or by initiating transitions between reactive and proactive inhibitory mechanisms. Future investigations might

profitably consider the distinction between reactive and proactive mechanisms when developing tasks to probe the fundamental function of pre-SMA. The research was funded by the UK Medical Research Council and a grant from the Wellcome Trust (098282). “
“How human infants map speech sounds to meaning in order to break into semantics is a key question for understanding the ontogenesis of language. It has been suggested that a biologically endowed ability to realize cross-modal mapping, particularly between auditory and visual percepts, scaffolds language learning in human infants (Imai et al., 2008 and Maurer et al., 2006). Consistent with this idea, 4-month-old infants appear to sense intrinsic correspondences between speech sounds and certain features of visual input (see Ozturk et al.

By convention the ONSD is assessed 3 mm behind the papilla. In order to gauge the ONSD, the distance between the external borders of the hyperechoic area surrounding the optic nerve should be quantified (Fig. 1). Several studies reproduced a high intra- and interobserver reliability of the sonographic ONSD assessment [6], [7] and [8]. However, data on normal values

vary Proteases inhibitor considerably, especially in former publications [9]. This may be explained by differing ultrasound equipments and their influence on sonographic findings and measurement criteria different from the ones stated above. Therefore, several authors emphasized the necessity of correctly used measuring points and clearly displayed optic nerve structures for reliable results

[10] and [11]. In our study on this topic, using above criteria, the mean ONSD was 5.4 ± 0.6 mm in healthy adults that matches closely with results derived from two MRI studies [7]. Rohr et al. found a value of 5.3 ± 0.6 mm in patients with mental disorders but without intracranial lesions or signs of elevated ICP [12]. Geeraerts et al. indicated a mean ONSD of 5.1 ± 0.5 mm in healthy volunteers [13]. Accordingly, a cadaver study illustrated a good correlation between the evaluation of the ONSD by MRI and transbulbar sonography. Despite the unfavorable angle between the course of the optic nerve and the insonation direction in transbulbar sonography Steinborn et al. observed an acceptable agreement between MRI and the sonographic approach [11]. These results have been verified in an investigation of sixty-five children, recently [10]. In comatose or sedated patients with intracranial click here bleeding and traumatic head injury sonographic ONSD evaluation has been proven to be feasible in predicting raised ICP [3], [14] and [15]. An MRI-based investigation confirmed this observation [13]. Geeraerts et al. found a mean ONSD of 6.3 ± 0.6 mm in brain injured adults using sonography [14]. By means of MRI they

indicated a mean ONSD of 6.3 ± 0.5 mm Methocarbamol [13]. The threshold of ONSD predicting an elevated ICP was proposed to be between 5.7 and 5.9 mm [3], [13], [14] and [15]. In a metaanalysis of six studies with data on a total of 231 patients with traumatic brain injury or intracranial hemorrhage the technique had a sensitivity of 90% and a specificity of 85% [16]. Furthermore, transbulbar ONSD assessment has been suggested for follow-up examinations of children with internal hydrocephalus and ventriculoperitoneal shunt systems [17]. Moreover, two sonographic investigations observed a correlation between the severity of acute mountain sickness and ONSD [18] and [19]. Only few results were published on the sonographic ONSD evaluation in idiopathic intracranial hypertension (IIH) [20]. One MRI based retrospective study described a mean ONSD of 6.5 ± 0.9 mm in patients suffering from IIH and quote a cut-off value for raised ICP of 5.8 mm [21].

Bacteria conjugated to pHrodo™ show a very low fluorescent signal at the neutral pH present on the cell buy Ion Channel Ligand Library surface, but emit a bright red fluorescence in the acidic environment of phago-lysosomes. This level of discrimination eliminates washing and quenching

steps that are necessary with other non pH-dependent indicators of bacterial uptake. Moreover the fOPA here described takes advantage of the introduction of specific markers of HL-60 differentiation to neutrophils, which allow keeping under control the variability of effector cells. The method was evaluated for sensitivity and specificity, by testing a panel of sera from mice immunized with different GBS glycoconjugate vaccines against polysaccharide Ia. kOPA titers were compared with fOPA titers, and a confocal microscopy analysis was conducted to study bacterial localization inside neutrophils, PARP inhibitor in the presence or in the absence of specific antibodies and

complement. GBS strains 515 (serotype Ia) (Baker et al., 1982) and COH1 (serotype III) (Wessels et al., 1992) were used in this work. Bacteria were grown in Todd–Hewitt Broth (THB) to an optical density at 600 nm (OD600 nm) of 0.45. Ten percent glycerol was added to the culture before dispensing 1 ml aliquots in cryo-vials for flash freezing in a 95% ethanol-dry ice bath. Frozen cultures were kept at − 70 °C until use. OPAs were performed with rabbit and mouse sera. Rabbit sera were raised by immunizing one animal with three doses of monovalent CRM197-conjugated polysaccharide Ia, Ib and III in presence of aluminum hydroxide (Alum). Mouse sera were pooled from animals immunized with a GBS vaccine composed by polysaccharide Ia, Ib and III conjugated to CRM197, formulated with Alum or MF59 (Podda, 2001). Animal treatments were performed in compliance with the Italian laws and approved by the institutional review board (Animal Ethical Committee) of Novartis Vaccines and Diagnostics, Siena, Italy. Bacteria were grown in THB

Dichloromethane dehalogenase to OD600 nm = 0.6, washed twice with Phosphate Buffered Saline (PBS, pH 7.2–7.4,Gibco) and suspended in half volume of PBS-0.08% paraformaldehyde (PFA, Sigma). Cells were incubated at 37 °C for 30 min and kept at 2–8 °C in PBS-0.08%PFA. Immediately before labeling, cells were washed with PBS, suspended at 20 mg (wet weight)/ml using a freshly prepared 100 mM Sodium Hydrogen Carbonate solution pH 8.5 (Merck) and split into aliquots of 750 μl. A 10 mM stock solution of PHrodo™ Succinimidyl Ester (Invitrogen) in dimethyl sulfoxide (Sigma) was diluted in the bacterial suspension at a final concentration of 0.1 mM. Each sample was incubated for 45 min at room temperature in the dark and then added with 750 μl of Hank’s Balanced Salt Solution with Ca2 + and Mg2 + (HBSS, Gibco), then spin down with a bench top centrifuge for 60 s at 14,000 ×g. The supernatant was aspirated and the pellet suspended in HBSS and stored in the dark at 4 °C for two months.