Tanja
Sauer
a,
Martin
Raithel
b,
Jürgen
Kressel
b,
Sonja
Muscat
a,
Gerald
Münch
c and
Monika
Pischetsrieder
*a
aDepartment of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, Friedrich-Alexander University, Schuhstr. 19, 91052, Erlangen, Germany. E-mail: monika.pischetsrieder@lmchemie.uni-erlangen.de
bFunctional Tissue Diagnostics, Gastroenterology, Department of Medicine I, Friedrich-Alexander University, Ulmenweg 18, 91054, Erlangen, Germany
cDepartment of Pharmacology, School of Medicine, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia
First published on 9th September 2011
In the healthy gut, NF-κB is a critical factor of the intestinal immune system, whereas inflammatory bowel diseases are associated with chronic activation of NF-κB. Previous studies indicated that coffee induces nuclear translocation of NF-κB in macrophages, an effect attributed to roasting products. In the present work, coffee extract or roasting products induced nuclear translocation of NF-κB in macrophages, Caco-2 cells, and primary human intestinal microvascular endothelial cells (up to fivefold, p < 0.001). Since the effect clearly depended on the cell type, ex vivo experiments were performed with intact human gut tissue from biopsies. The uniformity of the specimens and tissue viability during ex vivo incubation for up to 2 h were verified. Roasting products led to a concentration dependent significant increase of nuclear translocation of NF-κB in human gut tissue (up to 2.85 fold increase, p = 0.0321), whereas coffee extract induced a trend towards higher nuclear NF-κB concentration. NF-κB activation in macrophages and Caco-2 cells by roasting products was significantly blocked by co-incubation with catalase (p = 0.011 and p = 0.024) indicating involvement of H2O2-signaling. Monitoring of extracellular H2O2 indicated that roasting products in coffee constantly generate H2O2 by spontaneous oxygen reduction, which is only partially detoxified by cellular antioxidative systems. Thus, it can be concluded that ex vivo stimulation of intact human gut tissue is a valuable model to study nutritional effects on complex tissue systems. Furthermore, the consumption of coffee and roasting products may be able to induce nuclear NF-κB translocation in the human gut.
It has been shown that coffee and coffee components considerably influence the activation of NF-κBin vitro and in vivo. In unstimulated macrophages, the presence of coffee induced strong activation of NF-κB. This effect was attributed to hydrogen peroxide (H2O2)-generating melanoidins and Maillard products formed during the roasting process.10 In LPS-stimulated monocytes as well as in the liver and the kidney of transgenic reporter mice, a high concentration of coffee inhibited LPS-induced activation of NF-κB.11 Although the diterpenoid lipids kahweol and cafestol are able to reduce LPS-induced NF-κB activationin vitro, this effect was rather related to melanoidins formed during the roasting process.11,12 The goal of the present study was, therefore, to investigate the intestinal activation of NF-κB by coffee. For this purpose, the time-, concentration- and cell-type-dependent NF-κB activation was examined upon cell stimulation with coffee and roasting products. Furthermore, a model was established to investigate the influence of food components on intact human gut mucosa tissue ex vivo. The model was then applied to analyze the effects of coffee and melanoidins on NF-κB activation.
Fig. 1 Nuclear translocation of NF-κB in NR8383 macrophages (A/B) and Caco-2 cells (C) induced by coffee extract. Cells were stimulated with coffee extract (1–4 mg mL−1) or TNF-α as positive control for 0.5–6 h. The intensity of the p65 signal (NF-κB subunit) was related to the loading control β-actin and expressed as n-fold increase compared to water-treated control cells (bars). Cell viability was assured by trypan blue dye exclusion test (points). Data is mean ± SD (A: n = 3–5; B: n = 3; C: n = 2). * p < 0.05, ** p < 0.01, *** p < 0.001. Representative Western blot of p65 and β-actin in NR8383 (A/B) and Caco-2 cells (C). |
Cytokine release as a result of stimulating NR8383 macrophages was analyzed using the Bio-Plex system including interleukin (IL)-1α, IL-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α. The results indicate that under the tested conditions, roasting products do not exert pro-inflammatory activity (data not shown).
Fig. 2 Nuclear translocation of NF-κB by roasting products in various cell types. NR8383 macrophages, Caco-2 cells, and HIMEC were stimulated with different concentrations of MRM for 2 h. In case of catalase co-treatment, catalase (150 U mL−1) was added ten minutes prior to stimulation. The intensity of the p65 signal (NF-κB subunit) was related to the loading control β-actin and expressed as n-fold increase compared to PBS-treated control cells. Data is mean ± SD (n = 2–8); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control (a) or to the corresponding incubation without catalase addition (b). Representative Western blot of p65 and β-actin in NR8383 macrophages (A), Caco-2 cells (B) and HIMEC (C). |
Cell viability of the tissue cells during mucosa oxygenation ex vivo was investigated by the lactate dehydrogenase (LDH) assay. Intact human gut tissue samples were kept in modified PBS or Hanks buffer during mucosa oxygenation between 0.5 and 24 h. The amount of LDH secreted into the supernatant compared to overall LDH rose time-dependently in both media indicating a decrease in cell viability over time (Fig. 3). After 4.5 h, the cell viability still exceeded 75% in both media. However, after 6 and 24 h, cell viability decreased to about 50% and nearly 0%, respectively, independent from the cell culture medium. The difference in cell viability between both media was not significant. A similar LDH release was observed when a single human gut tissue sample was monitored between 0.5 and 24 h (data not shown). Therefore, a stimulation time of 2 h was chosen for further experiments to avoid notable cell death during the ex vivo incubation.
Fig. 3 Time-dependent cell viability of human gut tissue samples during mucosa oxygenation ex vivo. The secretion of LDH assay into the supernatant was used as indicator of cell death and thus cell viability. For each time point a separate human gut tissue sample was incubated in modified PBS or Hank's balanced salt solution buffer (Hanks). LDH concentration was measured in the supernatant and in the tissue. The cell viability was expressed as the ratio between LDH concentration in the supernatant and the overall LDH concentration. Mean ± SD (n = 2–3) is shown. |
Fig. 4 Nuclear translocation of NF-κB induced by roasting products in human gut tissue ex vivo. The tissue samples were stimulated with MRM (10–100 mM) in modified PBS for 2 h. The intensity of the p65 signal (NF-κB subunit) was related to the loading control β-actin and expressed as n-fold increase compared to PBS treated control. Data are mean ± SD (n = 5–7); * p < 0.05, ** p < 0.01, *** p < 0.001. Representative Western blot of p65 and β-actin in human gut tissue. |
The experiment was repeated with coffee extract as stimulant. Coffee extract induced a twofold higher NF-κB translocation compared to the control. The difference, however, was statistically not significant (Fig. 5).
Fig. 5 Nuclear translocation of NF-κB induced by coffee extracts in human gut tissue ex vivo. The specimens were stimulated with coffee extract (1–4 mg mL−1) in modified PBS for 2 h. The intensity of the p65 signal (NF-κB subunit) was related to the loading control β-actin and expressed as n-fold increase compared to control. Data are mean ± SD (n = 3–5). Representative Western blot of p65 and β-actin in human gut tissue. |
In order to clarify the role of H2O2 and of cellular H2O2-detoxification in MRM-induced NF-κB-translocation, NR8383 macrophages were incubated with MRM (10–100 mM) for up to 24 h and the extracellular H2O2 concentration was measured over time (Fig. 6A). In addition, H2O2 levels were analyzed in MRM stored at identical cell culture conditions, but in the absence of NR8383 macrophages (Fig. 6A). In the presence of NR8383, between 100 and 450 μM extracellular H2O2 was detected after 2 h depending on the MRM concentration. After 12 h, H2O2 levels were slightly decreased but not completely scavenged. In comparison, the overall H2O2 concentration was significantly higher in the absence of NR8383 macrophages than in the presence of cells. The H2O2 concentration in MRM without cells reached levels between 170 μM and 565 μM after 2 h dependent on the MRM concentration. Other than in the presence of cells, H2O2 generation was promoted with increasing incubation time.
Fig. 6 Extracellular H2O2 concentration after 2, 6, 12, and 24 h in NR8383 macrophages incubated with (A) MRM (10–100 mM) or (B) coffee extract (1–4 mg mL−1) for 24 h. Simultaneously, (A) MRM and (B) coffee extract were stored under similar cellular conditions but in the absence of NR8383 macrophages and H2O2 concentration was investigated likewise. Data is mean ± SD (A: n = 4; B: n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. |
Both experimental approaches were repeated under co-incubation of catalase (150 U mL−1). No significant amount of extracellular H2O2 was detected within the first 12 h (data not shown).
According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay, the cell viability of NR8383 macrophages was not impaired, but cell growth was rather enhanced after incubation with MRM in low concentration. After exposure to 25 mM MRM for 24 h, the cell viability amounted to 173 ± 23%. In this case, the supplementation of catalase did not show any significant impact on the cell viability of MRM treated NR8383 macrophages. However, 50 mM and 100 mM MRM possessed cytotoxic potency (59 ± 21% and 23 ± 7% cell viability), which was reduced by co-treatment with catalase (121 ± 15% and 42 ± 5% cell viability).
Fig. 7 Extracellular H2O2 concentration after 2 h in human gut tissue samples incubated with (A) MRM (10–100 mM) or (B) coffee extract (1–4 mg mL−1) during mucosa oxygenation ex vivo. Simultaneously, MRM and coffee extract were cultivated under the same conditions as aforementioned but in the absence of the tissue samples and H2O2 concentrations were investigated likewise. Data are mean ± SD (A: n = 3; B: n = 2–5); * p < 0.05, ** p < 0.01, *** p < 0.001. |
Coffee extract as well as roasting products produced in a MRM significantly increased the nuclear translocation of NF-κB in macrophages in a concentration- and time-dependent manner. In contrast, stimulation of the colorectal epithelial cells Caco-2 by different concentrations of coffee did not lead to increased nuclear translocation of NF-κB. The incubation of Caco-2 cells with roasting products led to a significantly increased NF-κB response, which was, however, considerably lower than the effect on macrophages. Additionally, the primary human intestinal cells HIMEC were treated with MRM, since it could be expected that the metabolism of immortalized cell lines differs from cellsin vivo. As a matter of fact, NF-κB responded not only in multiple cell lines, but also in primary intestinal cells, which show closer similarities to cell metabolismin vivo. MRMs were already shown to induce NF-κB translocation in macrophages as well as in kidney cells.10 Besides, individual Maillard products such as aminoreductones15 and glycated ovalbumin16 elevated nuclear NF-κB amounts in macrophages and dendritic cells, respectively. Thus, it can be concluded that roasting products induce nuclear translocation of NF-κB in various cell types. The degree of NF-κB activation, however, varied depending on the cell type and showed the maximum effect in macrophages. The NF-κB activation by coffee was lower under the applied experimental conditions and could only be detected in macrophages. Furthermore, it was reported that coffee and coffee roasting products protect from hepatic inflammatory processes induced by a high fat diet in mice.17
The in vitro experiments raised the question how NF-κB responds in human intestinal tissue consisting of a wide range of cell types. The mucosa layer of the gut comprises a variety of cell types including immune cells such as macrophages, which showed the most explicit NF-κB response. Particularly in case of IBD such as Crohn's disease, macrophages are increasingly infiltrated in the mucosa of the intestine. Not only IBD, but also food allergies are triggered by specific reactions of the gastrointestinal immune system after food intake. Therefore, the immunomodulatory effect of coffee extract and roasting products was studied in the mucosa of the gut as the largest immunological organ of the body. Hence, a method was successfully established which allowed the stimulation of human gut tissue ex vivo.18,19 It was guaranteed that the tissue samples per se were uniform and comparable to each other and that cell viability of the tissue samples was not harmfully affected by the mucosa oxygenation ex vivo. Whereas roasting products formed in MRM elevated nuclear NF-κB levels in gut tissue significantly, coffee extract showed a trend towards activation.
In agreement with the in vitro studies, both coffee and roasting products showed similar effects on nuclear NF-κB translocation in human gut tissue, but with differences in the level of activation. These differences may be attributed to a different concentration of active roasting products in coffee and MRM, but also to coffee ingredients which may counteract NF-κB activation such as kahweol and 3-methyl-1,2-cyclopentanedione.12
Two milligrams per millilitre of coffee extract, which induced strong nuclear NF-κB translocation in macrophages, corresponds to a 1:7 diluted coffee beverage. Assuming a conversion rate for ribose into roasting products of 30%, 50 mM MRM would correspond to the melanoidin content of a coffee beverage. As far as we know, the concentration of coffee components in the gut after coffee consumption is not clear. Therefore, further studies are required to investigate the effect of coffee and roasting products on NF-κB activationin vivo.
Furthermore, mechanisms were investigated which may explain how NF-κB activation in different cell types and complex human gut tissue can be differently affected by coffee and by roasting products. H2O2 seems to be a major factor of cell signaling induced by coffee and roasting products. H2O2 has been recognized as an important second messenger in redox signaling.20Co-treatment with catalase blocked the NF-κB activation in macrophages by the incubation with MRM or coffee extract, respectively.10 The present study revealed similar suppression for macrophages and Caco-2 cells. Since extracellular catalase cannot penetrate cellular membranes, its action is restricted to the decomposition of extracellular H2O2.21 It is well documented that coffee and roasting products are able to generate H2O2 in a cell-free system.10,22–24Extracellular H2O2 would then be able to penetrate the cell, activating H2O2-induced cell signaling including the activation of NF-κB. Indeed, supplements of pure H2O2 were shown to trigger the activation of NF-κB.25 Thus far, it is not fully clear to which extent coffee and roasting products are able to generate H2O2 in the anaerobic environment of the gastrointestinal tract, or if any H2O2 ingested with the coffee beverage reaches the gastrointestinal tract. For this purpose, studies are required to investigate the generation of H2O2 from roasting products in detail under different conditions. However, it has been shown that coffee drinking leads to increased urinary H2O2 concentrations in humans.26,27 These data strongly indicate continuous generation of H2O2 or high stability of coffee-derived H2O2in vivo. Furthermore, it has been demonstrated that other food components, such as green tea polyphenols, which are able to generate H2O2in vitro, are also able to generate reactive oxygen speciesin vivo. This prooxidative effect has been associated with the induction of apoptosis and the upregulation of antioxidative enzymes, eventually contributing to cancer preventive effects of tea polyphenols.28 The susceptibility of cells and tissue samples to nuclear NF-κB translocation depends on the activity of the stimulant to generate H2O2, but also on the ability of H2O2 to diffuse through cell membranes mediated by aquaporins.29 Furthermore, the cell-specific capacity of antioxidative enzymes such as glutathione peroxidase, peroxiredoxins, or catalase and numerous non-enzymatic antioxidants will have a major influence on NF-κB activation. The present study demonstrated that coffee and, particularly, roasting products are potent generators of H2O2 in different cell-free environments. In the presence of macrophages, H2O2 was clearly detectable in the extracellular space when exposed to coffee and roasting products, but the concentration was significantly lower than in the absence of cells. The decline of extracellular H2O2 can thus be attributed to diffusion across the cell membranes and also to cellular antioxidative systems. In C6 glioma cells, for instance, a bolus of H2O2 (≤100 μM) was completely detoxified.30 But in contrast to a one-time bolus of pure H2O2, roasting products in coffee and MRM generate H2O2 permanently, which could be responsible for the induction of nuclear NF-κB translocation in the different cell types.
In a similar way, the human gut tissue was able to significantly reduce extracellular H2O2 produced by coffee or roasting products. However, the detoxification rate of the tissue was higher compared to macrophages. The higher detoxification rate can be caused by the diversity of cells present in the tissue samples, but also by a higher cell density. Thus, the minor amount of residual H2O2 in the tissue samples may cause a reduced NF-κB response compared to macrophages. Finally, different gut sections may be differently affected by coffee roasting products. Whereas the upper gut section is directly exposed to roasting products, biotransformation processes and resorption may modulate the chemical structure or decrease the concentration of the roasting products reaching the lower gut resulting in changes of their bioactivity. The biological consequences of coffee-mediated NF-κB activation are still not fully clear. A screening assay for cytokine release indicated that NF-κB translocation induced by roasting products is not connected to pro-inflammatory activity, which is in agreement with previous studies.10 Active NF-κB regulated the expression of more than 150 target genes, including, for example, genes encoding immunoreceptors, cell adhesion molecule, stress response, cell-surface receptors, regulators of apoptosis, or enzymes.1 Therefore, large arrays for expression analysis have to be applied to delineate the full cellular response to intestinal NF-κB activation mediated by roasting products. Furthermore, the response of inflamed tissue, as present, for example, in IBD, may greatly differ from the response of unstimulated tissue.
It can be hypothesized that in a healthy gut, coffee-induced stimulation of NF-κB signaling supports the maintenance of immune homeostasis, particularly in the epithelial tissue, thus contributing to a balanced gut health.31 This assumption is in accordance with a moderate favorable effect of coffee drinking on colorectal cancer.32 In patients suffering from IBD, however, further activation of NF-κB by coffee may worsen inflammation processes associated with sustained elevated NF-κB activation.31
More specifically, NR8383 macrophages (3 × 106cells) were grown for 4 d. Thereafter, cells were incubated with the stimulants in PBS for up to 6 h. In order to replace cell culture medium by PBS, floating cells were collected by centrifugation (1500 rpm, 2 min, at room temperature) and both floating cells and adherent cells were washed separately with PBS. Finally, the floating cells were re-suspended in PBS and recombined with the adherent cells.
Caco-2 cells (1 × 106cells) and HIMEC (7.5 × 105cells) were grown for 5 d. In the case of HIMEC, the medium was refreshed on the 4th day. On the 5th day, the medium was removed for both, adherent cells were washed with PBS and cells were stimulated in PBS for 2 h.
After stimulation, the nuclear cell extracts were prepared on ice according to Andrews and Faller with slight modifications.34 Briefly, floating cells (NR8383) were collected by centrifugation (1500 rpm, 4 min, 4 °C); adherent cells by scraping. Floating and adherent cells were merged and washed three times with ice-cold PBS. Next, the cells were lyzed with 1 mL ice-cold hypotonic buffer Acell (10 mM HEPES, 10 mM KCl, 1 mM MgCl2·6H2O, 5% (v/v) glycerol, 0.5 mM EDTA, 0.1 mM EGTA), which was supplemented with protease inhibitor solution (Protease Inhibitor tablet complete (PIS) was dissolved in 0.5 mL PBS; 1% (v/v)), PMSF (2 mM in ethanol), and DTT (0.5 mM in water). After 15 min incubation on ice, 65 μL of 10% (v/v) NP-40 (in water) was added and the cells were mechanically lyzed by vortexing for 15 s. Cell nuclei were collected by centrifugation and washed with buffer Acell to ensure a complete removal of cytoplasmic proteins. Finally, nuclear proteins were extracted for 1 h with 52 μL ice-cold high salt extraction buffer B (20 mM HEPES, 1% (v/v) NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2·6H2O, 20% (v/v) glycerol, 0.5 mM EDTA, 0.1 mM EGTA), which was supplemented with PIS (1%, v/v), PMSF (2 mM), and DTT (0.5 mM). The suspension was vortexed every 20 min. After 1 h, the supernatant nuclear extract was collected after centrifugation. The protein concentration of the nuclear extract was determined with the Dc-protein assay using BSA in high salt extraction buffer B as standard. Nuclear cell extracts were stored at −80 °C until use for Western blotting.
After stimulation, nuclear proteins were extracted according to a modified method of Thiele et al.35 In detail, intestinal human gut tissue samples were washed with PBS in a 50 mL Falcon tube for 2 min at 400 rpm. Cells were lyzed by vortexing in ice-cold hypotonic buffer Atissue (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl), which was freshly supplemented with DTT (0.5 mM), PMSF (0.2 mM), PIS (1% (v/v)), and NP-40 (0.56% (v/v)). To ensure lysis, the gut tissue was subjected to three freeze-thaw cycles in liquid nitrogen and finally mechanically disrupted in the Ultra Turrax homogenizer for 1 min on ice. After 30 min, the cell nuclei were collected by centrifugation and washed with buffer Atissue to assure the complete removal of cytoplasmic proteins. Subsequently, the nuclear proteins were extracted from the nuclei with 25 μL of high salt extraction buffer B.
Briefly, nuclear proteins were denatured in loading buffer (0.06 M Tris-HCl, 0.07 M SDS, 1.04 M urea, 0.064 M DTT, 7% (v/v) glycerol, bromophenol blue, adjusted to pH 7.4 with 2 N NaOH) for 7 min at 95 °C. Equal amounts of denatured protein (10 μg for cell experiments; 5 μg for tissue experiments) were separated by electrophoresis in a 12% SDS-polyacrylamide gel and afterwards transferred to a nitrocellulose membrane. The membranes were cut into two parts and the non-specific binding sites of the membrane were blocked with blocking buffer (0.15% (w/v) skim milk powder in PBS/Tween (0.1% (v/v) Tween 20 in PBS). Then the membranes were incubated overnight at 4 °C with the primary antibodies for p65, a subunit of NF-κB, or for β-actin as loading control. Primary antibodies included rabbit polyclonal anti-p65 (diluted 1:500 in blocking buffer) or, in the case of Caco-2 cells, mouse monoclonal anti-p65 (diluted 1:100 in blocking buffer) and mouse anti-β-actin (diluted 1:13333 in blocking buffer). Next, the membranes were incubated for 1 h with the HRP-conjugated second antibody (anti-rabbit 1:1500 diluted in blocking buffer; anti-mouse 1:2000 diluted in blocking buffer). The protein bands were visualized by ECL. The p65 band was identified by an anti-p65 antibody blocking peptide and a chemiblot™ molecular weight marker. The data was analyzed densitometrically by a VersaDoc™ imaging system. Besides the adjusted protein amount, the intensity of the p65 (NF-κB) signal was normalized to the loading control β-actin. Similar results were obtained when the NF-κB signal was not normalized to β-actin. NF-βB translocation was expressed as n-fold increase relative to the control exposed to the solvents PBS/water instead of the stimulants. The identity of p65 was verified by the use of a p65 blocking peptide.
Human gut tissue samples were kept either in incubation media, modified Hank's balanced salt solution (Hanks) (3 g L−1 albumin, HEPES (1 M) 2.4% (v/v), FCS 1% (v/v)) or modified PBS (3 g L−1 albumin, HEPES (1 M) 2.4% (v/v)). LDH was measured after 0.5, 1.5, 3, 4.5, 6, and 24 h in the supernatant and in the tissue. A separate biopsy was used for each time point. Aliquots of the supernatant were used to determine the LDH release into the medium. In order to analyze the intracellular LDH amount, the human gut tissue samples were homogenized in an Ultra Turrax in a defined volume of bis-tris buffer (20 mM; pH 7) on ice for 30 s. The LDH concentration was quantified according to a protocol of Beckman Coulter. Cell death is defined by the LDH release, which is calculated as the percentage of extracellular LDH related to the total LDH amount. The cell death values were finally used to calculate the cell viability.
In order to rule out any influence of the human gut tissue samples themselves, the kinetic LDH release was analyzed for one single biopsy over time for 24 h. For that purpose a biopsy was incubated in a modified Hanks buffer for 24 h. Aliquots of the supernatant were taken after several time points and the extracellular LDH amount was determined as described above.
NF | Nuclear factor |
LPS | Lipopolysaccharide |
IBD | Inflammatory bowel disease |
Caco-2 cells | Human epithelial colorectal adenocarcinoma cells |
MRM | Maillard reaction mixture |
IL | Interleukin |
TNF | Tumor necrosis factor |
PBS | Phosphate buffered saline |
HIMEC | Human intestinal microvascular endothelial cells |
LDH | Lactate dehydrogenase |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide |
PMSF | Phenylmethylsulfonyl fluoride |
DTT | Dithiothreitol |
SDS | Sodium dodecyl sulfate |
BSA | Bovine serum albumin |
HRP | Horseradish peroxidase |
PCA | Perchloric acid |
HEPES | 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid |
BC | Bicinchoninic acid |
ECL | Enhanced chemiluminescence |
DMSO | Dimethyl sulfoxide |
FCS | Fetal calf serum |
MEM | Minimum essential medium |
PIS | Protease inhibitor solution |
FOX | Ferrous oxidation xylenol orange |
This journal is © The Royal Society of Chemistry 2011 |