Human umbilical vein endothelial cells (HUVEC) (Lot#0000120825; Lonza®, Walkersville, MD, USA) were cultured at 37 °C and 5% CO2 in endothelial basal media (EBM-2) supplemented with a bullet kit (Lonza®) containing human fibroblast growth factor B, hydrocortisone, vascular endothelial growth factor, ascorbic acid, heparin, human

click here endothelial growth factor, and fetal bovine serum. For cell passage, cultures were incubated to approximately 40% confluence within the culture flask, according to LONZA guidelines. For experiments, cultures were incubated to approximately 50% confluence then harvested by exposure to trypsin–EDTA (Lonza®) for 2 min at 37 °C. Cell suspensions were centrifuged at 201g for 5 min in a 5810R tabletop centrifuge (Eppendorf,

Westbury, NY, USA), and resuspended in endothelial growth media at a concentration of 1.0 × 106 cells/mL in 1 mL aliquots maintained in 12 × 75 mm round bottom plastic tubes (VWR, Edmonton Canada) prior to experimentation. The dual fluorescent assay (SytoEB) 5-FU nmr uses a combination of two fluorescent dyes, Syto13 (Molecular Probes, Eugene, OR, USA) and ethidium bromide (EB) (Sigma–Aldrich, Mississauga, ON, Canada) to assess cell membrane integrity. Syto13 is a DNA/RNA binding stain that permeates all cells and fluoresces green on excitation by UV wavelengths. Ethidium bromide permeates cells with damaged plasma membranes, exhibiting red fluorescence upon UV exposure. The combination of these two dyes makes a binary assay with membrane intact cells exhibiting green fluorescence (Syto13) and membrane compromised cells exhibiting red fluorescence (EB). The SytoEB stain was prepared using 1× phosphate buffered saline (PBS), and aliquots of Syto and EB diluted from the stock solution. The final dye was comprised of 25 μM EB and 12.5 μM Syto13. 10 μL of the prepared dye were added to the 1 mL aliquot of HUVEC in suspension and incubated for 2 min at room temperature before analysis. The ratiometric dye 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine-iodide

(JC-1) (Molecular Probes, Eugene, OR, USA) was used as an indicator of mitochondrial membrane potential Glycogen branching enzyme of HUVEC in suspension. The fluorescence shifts from green (∼525 nm) in low polarization states (non-functional mitochondria) to red (∼590 nm) in high polarization states (functioning mitochondria). This change in color of fluorescence is based on a concentration-dependent shift from monomers of the dye which fluoresce green to J-aggregates which fluoresce red [34]. Initially the dye is present as cationic monomers (green) that permeate into cells, influenced by the negative intracellular potential. In healthy cells these monomers permeate into the mitochondrial matrix, drawn by the electronegative interior of mitochondria where these monomers form J-aggregates (red) [15].

It has been shown that the pro-inflammatory cytokine tumour necrosis factor-α (TNFα) induces rapid phosphorylation of IκBα and its ubiquitin-induced degradation. This event is necessary for NFκB/p65 to be released from the complex with IκBα and for its relocation to the nucleus where it exerts transactivation functions by binding co-activator proteins [reviewed in [39]]. As

incubation with PCP resulted in decreased phosphorylation of IKKβ-mediated phosphorylation of NFκB/p65 at the activating S536, we addressed the question whether PCP affected the TNFα-mediated translocation of NFκB/p65 into the nucleus. As shown in Fig. Epigenetics Compound Library high throughput 7, treatment of both cell lines with TNFα led to the accumulation of NFκB/p65 in the nucleus with respect to DMSO- and PCP-treated cells, respectively, where NFκB/p65 appeared

to localize in the cytoplasm. However, incubation of cells with PCP suppressed TNFα-induced migration of NFκB/p65 into the nucleus as indicated by the persistent signal in the cytoplasm. The study presented here, indicates that PCP is the active component of C11 exerting cytotoxic effects in human pancreatic cancer Selleckchem STA-9090 cell lines. Our previous studies showed that simultaneous silencing of the CK2 catalytic subunits by RNA interference enhances the sensitivity of these cell lines towards chemotherapeutic agents currently used in the clinics for the cure of advanced pancreatic cancer [[5], for a review see [40]]. We show here that PCP inhibits recombinant human CK2 in an ATP-competitive manner as well as the endogenously expressed enzyme as revealed by the decreased phosphorylation of the molecular chaperone Cdc37 at S13, a known cellular substrate target of CK2 [Fig. 6., [38]]. Evidence indicates that CK2 supports survival and confers resistance to chemotherapeutic treatment of cancer cells [reviewed in [2] and [6]]. Hence, PCP-mediated induction of cell death in human pancreatic cancer cells reported here, may be due, at least partially, to the inhibition of endogenous

CK2. However, as protein kinase CK2 expression levels have been shown to be elevated in cancer and highly proliferating cells, we cannot exclude that other types of cancer cells would respond to PCP treatment in a similar fashion. for The poor prognosis of pancreatic cancer is in part attributed to the presence of a subgroup of cancer stem cells which account for tumour recurrence due to their self-renewal, metastatic potential and resistance to cytotoxic drug treatment [19] and [20]. Interestingly, we show here that incubation of a sub-population of Panc-1 cells enriched in cancer stem cells with PCP induces a level of cytotoxicity comparable to the one observed in the cancer stem cells-depleted population suggesting that PCP treatment lowers the intrinsic resistance of cancer stem cells to cell death induction (Fig. 2).

Furthermore,

the cooking process did not significantly affect the BG content for either cultivation method, and this was reported previously ( Rungapamestry et al., 2006 and Verkerk et al., 2009). Among the analyzed vegetables, watercress behaved differently. No significant difference in benzylglucosinolate content was observed between the organically and conventionally cultivated plants. Among the other analyzed Brassicaceaes, organic collard greens had the highest BG content ( Fig. 2). In conclusion, the organic cultivation practice led to increased concentrations of total glucosinolates and benzylglucosinolate in most of the vegetables. These differences were more apparent when the compounds were isolated and separated using HPLC high resolution liquid chromatography. The acidified methanol extraction of broccoli tissues resulted in significantly higher levels of GLs, which selleckchem differentiated the two modes of cultivation. This difference was supported by the chromatographic analysis of benzylglucosinolate. The tissue extract analysis without the addition of TFA revealed the same concentration profile, but the concentrations of compounds were much lower. Among the evaluated Brassicaceaes, watercress exhibited a different profile for benzylglucosinolate and GL concentration;

significantly higher concentrations of the compounds were Selleckchem PF-562271 observed in conventionally cultivated watercress. These results suggest that watercress cultivated conventionally is tuclazepam more efficient at sulfur absorption. The highest levels of glucosinolates and benzylglucosinolate were found in Brassica cabbage and broccoli. Furthermore, cooking significantly decreased the GL content of vegetables, but the more accurate HPLC analysis showed that the benzylglucosinolate profile was unaffected. Thus, we believe that these types of plants, if cultivated organically, may become promising sources of secondary metabolites and may reveal gene targets that could confer resistance against phytopathogenic pests and diseases of agro-economic importance; this would contribute to environmental sustainability

without the use of radical agricultural production systems. The authors thank FAPESPand CNPq for supporting this work. We also thank Beatriz Rosana Cordenunsi and Eduardo Purgatto (Laboratory of Food Science Faculty of Pharmaceutical Science-University of São Paulo – SP/Brazil) for assistance with HPLC analyses. “
“A significant proportion of patients undergoing renal replacement therapy (RRT) with hemodialysis (HD) or peritoneal dialysis present a microinflammatory state, which is clinically detected by increased levels of C-reactive protein (CRP) and other inflammatory markers, mainly interleukin 1 and interleukin 6 [1] and [2]. This proinflammatory state is predictive of higher mortality levels and is associated with the malnutrition, inflammation, and atherosclerosis syndrome [3] and other factors, including the dialysis treatment itself [4], [5] and [6].

02 g/100 g) on TBARS values in mortadella (formulated with 150 mg/kg nitrite) stored for 24 days in packages with different atmospheres. The authors found lower lipid oxidation www.selleckchem.com/products/ipilimumab.html rates in samples with added EOs compared with controls. The use of 100 mg/kg nitrite without savory EO had the same effect on lipid oxidation as use of more than 7.80 μl EO in samples without nitrite. However, the use of 200 mg/kg nitrite and savory EO resulted in no positive effect on

lipid oxidation. Moreover, an antagonistic effect was observed in samples with 15.60 and 31.25 μl/g EO. This antagonistic effect suggests a possible interaction between nitrite and chemical compounds present in the fraction of S. montana Copanlisib nmr EO. The phenolic compounds might interact with nitrite by linking portions of the aromatic ring, and the antagonism might impair the antioxidant effect of the EO and nitrite. The use of natural additives has attracted attention, and some authors report that natural compounds have antioxidant effects similar to or better than those of synthetic preservatives. Sebranek, Sewalt, Robbins, and Houser (2005) compared

the antioxidant activity of rosemary extracts with the synthetic antioxidants BHA/BHT in sausages, using the TBARS method. The authors found that the natural and synthetic products yielded similar results. The interaction between the effects (essential oil concentration × nitrite levels × storage time) was significant (p ≤ 0.05) for the color coordinates lightness (L*), redness (a*), yellowness (b*), chroma (C*) and hue angle (h*). Values of CIE L* (lightness) for all treatments throughout the storage period are depicted in Fig. 3. Despite some differences during storage, in the samples with no nitrite, the addition of EO had no effects (p > 0.05) in lightness of mortadella. N-acetylglucosamine-1-phosphate transferase However, in samples manufactured with nitrite, the addition of EO at 31.25 μl/g affected lightness, which was significantly different from other treatments (p ≤ 0.05); this effect was most noticeable in samples with 200 mg/kg added. The effects of EO were dependent on the amount of nitrite added; the samples with 100 mg/kg nitrite were darkest (lower L*

values) at the end of storage, and those with 200 mg/kg nitrite had higher L* values throughout the storage period. This result is not in accordance with Hernández-Hernández et al. (2009), who observed a higher and negative correlation between lightness and TBARS values in model raw pork batters manufactured without nitrite; as oxidation increased (as TBARS), lightness decreased (the samples became darker). In this study, no relationship was found between TBARS values and lightness, not even in the samples without nitrite and savory EO. These results suggest that the darkening or lightening of cured cooked meat is not only related to lipid oxidation (and TBARS values) but also depends on an interaction between nitrite and certain EO compounds.

Because the NIH ‘Public Access’ policy is voluntary, authors may elect not to deposit such articles in PMC. If you wish to ‘opt out’ and not deposit to PMC, you may indicate this by sending an e-mail to [email protected]. There will be no need for you to post your manuscript Androgen Receptor Antagonist molecular weight directly to PubMed Central, and any such posting is prohibited. Individual modifications to this general policy may apply to some Elsevier journals and to its society publishing partners. GASTROINTESTINAL ENDOSCOPY will consider the following types of submissions. Authors should consider these categories and review recent issues of the journal

when preparing submissions. If you believe that your article should exceed

these word lengths, please contact Managing Editor Deborah Bowman at [email protected] and explain the reasons for the longer length. Word count does NOT include the abstract, tables, figure legends, take-home buy IWR-1 message, or references. • Original Article: work limited to 3500 words and 50 references reporting basic science or clinical investigations in areas relevant to gastrointestinal endoscopy. Original submissions will be considered for publication with the understanding that they are contributed solely to Gastrointestinal Endoscopy. If any material related to the submission (other than a brief abstract) has been published in any medium or has been submitted for publication elsewhere, the authors should provide copies of all related manuscripts, and outline the relationship of all materials for the Editor, to avoid allegations of duplicate publication. At the time an article is accepted and sent to Elsevier for production, a Journal Publishing Agreement will be e-mailed to the corresponding author. This Decitabine molecular weight original document, containing the author(s) ink signatures,

should be returned to Elsevier at the following address. This must be on file before publication can occur. Pushpa Vairam Elsevier, Inc. 360 Park Avenue South New York, NY 10010 E-mail: [email protected] Fax: 31-2048-52789 • The Journal Publishing Agreement must be completed in its entirety. Under Enter Classifications, authors must choose as many classifications as is appropriate for the article. Editors and reviewers will be assigned based on the classifications chosen. When prompted by the online submission process, authors should provide no fewer than three but no more than five key words that reflect the content of the manuscript. For guidance, consult the Medical Subject Headings (MeSH terms), available online at http://www.nlm.nih.gov/mesh/meshhome.html. The online instructions will guide you in creating this item.

Control wells contained (1) bacteria, peptone and antibiotic [streptomycin

(100 μg/mL) and ampicillin (80 μg/mL)]; (2) bacteria and peptone; and/or (3) peptone alone. Bacteria were grown in 20 mL tryptone soy buffer (TSB) with shaking for 17 h at 30 °C and then 100 μL of the E. coli k12 solution was transferred to 10 mL of TSB and incubated for a further 4 h. The bacteria were then Dabrafenib washed in PBS and diluted in TSB to a final concentration of 1 × 105 cells/mL. Fifty microliters of midgut sample were then incubated with 10 μL of bacterial suspension in triplicate in the wells of a sterile flat-bottom, 96-well microtiter plate (Nunc, Fisher Scientific UK, Leicestershire, UK). The optical densities were measured at 550 nm (OD550) at 37 °C and read at hour intervals from time zero for 12 h. All data points were subsequently blanked against time zero to account

for the opacity of the midgut samples and then the E. coli k12 readings were subtracted from all sample readings and multiplied by 100. Samples for the nitrite and nitrate determinations were collected in the same manner as for the antibacterial assays. The anterior midgut samples dissected nine days after feeding were homogenized in a tube with 200 μL of Milli-Q water and centrifuged at 8000 g for 1 min at 4 °C. Aliquots of 10 μL from supernatant Erlotinib ic50 were diluted in 90 μL of Milli-Q water. Nitrate and nitrite contents of samples were determined following the manufacturer’s instructions using the Griess Reagent System Assay Kit (Promega, WI, Methocarbamol USA), and absorbance of the product was measured at 550 nm (Moncada, 1992). Nitrite and nitrate contents were quantified as μmoles using a range of sodium nitrate standards and the specific activity was calculated as mg/mL of protein concentration in the anterior midgut samples. Protein content of samples was quantified with a protein assay kit (BCA∗ Protein Assay Reagent,

Pierce, USA) using bovine serum albumin (BSA) standards. The results were analyzed with GraphPad Prism 5 using 1 Way ANOVA or unpaired T test, or Mann Whitney test (nonparametric test) depending on the data distribution and number of treatments. Data were reported as mean ± standard error (SE) or as individual values with medians for parasite and microbiota populations. Differences among groups were considered not statistically significant when p > 0.05. Probability levels are specified in the text and figure legends. The physalin B treatment by oral, topical and contact application did not alter the physiology of the insects even when the insects were challenged by T. cruzi Dm28c clone. The mortality of all treated insects (around 9.6%) was similar to control (8.2%) during the 30 days and no alterations in the ecdysis process were observed. Experiments to investigate the direct effects of physalin B on T.

An east-west profile MK-1775 supplier direction was chosen in accordance with western Baltic herring spawning migration patterns in Lithuanian waters (Fedotova 2010). The patchiness of the spawning beds was substantial. We therefore assumed that small-scale bottom geomorphological features were important factors shaping the distribution of spawning beds and could affect these within a 100 m radius around the detected spawning location. Therefore,

for each point where spawning was recorded, 200 m long bottom surface profiles were drawn eastwards and westwards using a IVS3D Fledermaus 7 profiling tool with the spawning location in the middle. As a result we obtained two 100 m bottom surface profiles for each spawning location: towards the coast (eastwards) and towards the open sea (westwards). The bottom slope was estimated from these profiles, since it is an important geomorphological descriptor describing surface

steepness and direction. The slope is calculated as the ratio of the vertical (elevation) difference to the horizontal range. In this study bottom slopes were derived in two ways: as an average along the whole 100 m profiles and as an average along 10 m segments in order to indicate more local effects. Because of Daporinad price the direction of the bottom profiles, negative slope values correspond to western slopes (from west to east) and positive values correspond to eastern slopes (from east to west). For many fish species spawning is triggered by water temperature. Owing to the variety of spawning strategies of the Baltic herring this temperature is different for different populations (Elmer 1983, Evtjukhova & Berzinsh 1983, Aneer 1989, Kornilovs 1994, Klinkhardt 1996, Krasovskaya 2002). Baltic herring spawning usually starts in March (sometimes in February) in the south-western

and southern areas of the Baltic Sea when the water temperature reaches 4°C (Klinkhardt 1996), continues till the middle of summer and finishes in the northern parts at water temperatures up to 15–16°C (Krasovskaya 2002). Generally, spawning temperatures tend to Silibinin increase with latitude, resulting in later spawning seasons for the northern populations (Jørgensen et al. 2005). Water surface temperature was measured daily by the Lithuanian Environmental Protection Agency, Marine Research Department (EPA-MRD) at the Palanga meteorological station, which is located close to the centre of the multibeam area (Figure 1). The first eggs were found when the surface water temperature was 5.9°C in 2009 and 6.4°C in 2010 (Figure 4); in our area, therefore, spawning most likely begins when the temperature reaches approximately 6°C. These values are in good agreement with the general trend of herring spawning temperatures along the Baltic Sea (Krasovskaya 2002, Jørgensen et al. 2005). Baltic herring eggs were found at 25 sites, 18 of them located within the multibeam area (Figure 1). The majority (80%) of eggs were found within a depth interval from 4 to 8 m, with a mean depth of 6.5 ± 1.

The atmospheric model COSMO-CLM is a non-hydrostatic regional climate model. The model setup complies with CORDEX-EU in the CORDEX framework (Coordinated Regional climate Downscaling Experiment) (Giorgi et al. 2006). The domain covers the whole of Europe, North

Africa, the Atlantic Ocean and the Mediterranean Sea (Figure 1a). The horizontal resolution is 0.44° (approximately 50 km) and the time step is 240 seconds; it has 40 vertical levels. COSMO-CLM selleck products applies a ‘mixed’ advection scheme, in which a positive-definite advection scheme is used to approximate the horizontal advection while vertical advection and diffusion are calculated with a partially implicit Crank-Nicholson scheme. In COSMO-CLM, several turbulence schemes are available; in our experiments, we used the so-called 1-D TKE-based diagnostic closure, which is a prognostic

turbulent kinetic energy (TKE) scheme. It includes the interaction of air with solid objects at the surface (roughness elements). We modified the model code to adapt it to the coupled mode. Originally, COSMO-CLM did not have sub-grid scale ice; a grid over the ocean is either fully covered with ice or fully open-water. Thus, a grid size of 50 × 50 km2 implies a rather coarse approximation of real ocean conditions. In addition, COSMO-CLM does not have an ice mask over the ocean; an ocean grid is handled as sea ice or open water depending on the SST. If the temperature is below the freezing point of water, which is −1.7 °C Dasatinib molecular weight in COSMO-CLM, the surface is considered to be sea ice. When the temperature is equal to or higher than the freezing point, COSMO-CLM Cediranib (AZD2171) handles the surface as open water. However, a freezing point of water of −1.7 °C is applicable to sea water with a salinity of approximately 35 PSU

(Practical Salinity Units). In contrast, brackish sea water like the Baltic Sea has a much lower salinity than the average salinity of the World Ocean. At the centre of the Baltic Sea, the Baltic Proper, the salinity is only 7–8 PSU, and this decreases even further northwards to the Bothnian Sea, Bothnian Bay and Gulf of Riga (Gustafsson 1997). The freezing point of this brackish water should therefore be higher than −1.7 °C. When the freezing point is so low, the sea ice cover in the Baltic Sea in COSMO-CLM will be substantially underestimated. Therefore, when coupling COSMO-CLM with the ocean model NEMO, the sea ice treatment is modified in the surface roughness and surface albedo schemes. In the current albedo calculation scheme, COSMO-CLM attributes fixed albedo values to the water surface (0.07) and the sea ice surface (0.7) for the whole grid cell. In the coupled mode, as COSMO-CLM receives the ice mask from NEMO, it can now calculate the weighted average of the albedo based on the fraction of ice and open water in a grid cell. The surface roughness length of the sea ice and open-water grid is calculated in the turbulence scheme of COSMO-CLM.

9). The use of CHO-ldlD cells for flow cytometric analysis is complicated by the culture characteristics of the CHO-ldlD cells and therefore needed optimization. Since the CHO-ldlD cells scavenge the medium for free Gal and GalNAc, they must be cultured at low serum concentrations, to preserve the glycosylation defect. Additionally, because CHO-ldlD cells are adherent, the generation of a single cell suspension is accompanied by cell death. Dead cells are responsible

for a specific binding of antibodies and co-staining with 7AAD showed that in the 7AAD positive population there is a subpopulation learn more clearly positive for the MUC1 antibodies ( Fig. 3A). Moreover, reactivity with isotype antibody control could be detected, confirming the aspecific staining of the 7AAD positive cells. To decrease the number of dead cells and increase the yield, two different harvesting techniques were evaluated. Cell scraping was compared with trypsinization of the CHO-ldlD MUC1 cells. In contrast to cell scraping, trypsinization reduced the number of dead cells www.selleckchem.com/screening/inhibitor-library.html by 30%, however flow cytometric analysis showed that trypsinization induced expression of new epitiopes

reactive with the MUC1 specific antibodies, but not isotype control antibody ( Fig. 3B). Cleaving of the MUC1 peptide by trypsin could be responsible for this new epitope formation. Even though cell scraping induces a lot of cell death, it remains Rucaparib purchase the preferred option in this system since dead cells can be excluded using 7AAD staining. As shown in Fig. 2, the CHO-ldlD MUC1 system is effective in generating MUC1-Tn epitopes. To analyse if MUC1-Tn antibodies are present in sera of breast cancer patients as well as in healthy controls, CHO-ldlD MUC1 cells were cultured in the presence or absence of GalNAc and prior to flowcytometric analysis

cells were incubated with human serum. The CHO-ldlD cells were taken along as a negative control, to exclude aspecific or specific reactivity. Secondary antibody staining was performed for detection of serum antibodies to MUC1 and MUC1-Tn. Both anti-human IgM- and IgG-detecting secondary antibodies were used to discriminate between primary (IgM) and secondary humoral responses (IgG). Healthy controls as well as breast cancer patients did not show specific binding of serum antibodies with CHO-ldlD MUC1 cells cultured with or without GalNAc. Eventhough in the serum of breast cancer patients repetitively a marginal shift of the histogram could be observed when a secondary IgM-recognizing antibody was used, this shift did not reach significance ( Fig. 4A).

Since unconventional drilling is significantly different than conventional drilling, New York has been in the process of developing supplemental regulations (Supplemental Generic Environmental Impact Statement, SGEIS) which are pending the approval of the NYSDEC as of May 2014 (NYSDEC, 2013). Most county residents obtain their drinking water from groundwater, with residents in the major river valleys generally ABT-199 manufacturer tapping the glaciofluvial sand and gravel aquifers, in which, some aquifers are confined. Residents in the uplands primarily tap into bedrock aquifers (McPherson, 1993). In late 2011, Cornell Cooperative Extension collaborators placed newspaper ads in Chenango County newspapers

to recruit residents who would allow us to obtain samples from their water wells in exchange for receipt of a free water quality report. Interested county residents who responded to the ad were accepted into the study; only drilled wells as opposed to dug wells

or springs were included in this analysis. The 113 wells included in this analysis were distributed across the county (Fig. 2). Water samples were obtained from each of these homeowner wells between March and June 2012. The samples were taken from the closest accessible location to the well, which was often a spigot just past the water pressure tank in the basement. Water collection also occurred prior to the treatment system, if there was one. Water was initially Dasatinib price run to purge the pipes and pressure tank of stagnant water, for at least five minutes. A one liter pre-cleaned amber glass bottle was filled with water to be used for sediment and solute analysis. A second water sample was then taken for dissolved

gas analysis per standard methods of the USGS Reston Dissolved Gas Laboratory (Busenberg et al., 1998). For this method, flexible Masterflex Tygon tubing was attached to the spigot using a hose connector and water was run into a large bucket. The tubing was then inserted to the bottom of a 125 mL glass serum bottle and the bottle filled with water. With the water still running, the bottle was lowered into the bucket and then the tube was removed. After making sure no bubbles were adhering to the P-type ATPase inside of the bottle, a butyl rubber stopper was inserted in the bottle neck. A syringe needle was then inserted into the stopper that allowed the stopper to fully seal the bottle without having any remaining headspace. After sealing each bottle, the needle was removed, the bottle was removed from the full bucket, and the labeled sample bottles were stored in a cooler. Upon return to the Cornell Soil and Water Lab, a subsample of water for anion and cation analysis was removed from the amber collection bottle after ensuring it was well-mixed. The subsample was filtered to 0.45 μm and all samples were stored at 4 °C until analysis.