Continuous Density Gradient Research Articles

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

Comparison of Purification Solutions With Different Osmolality for Porcine Islet Purification.

The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank's balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient.

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a "bent-tipped" stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

An efficient method for isolation of representative and contamination-free population of blood platelets for proteomic studies

To date, there has been no ideal method for blood platelet isolation which allows one to obtain a preparation devoid of contaminations, reflecting the activation status and morphological features of circulating platelets. To address these requirements, we have developed a method which combines the continuous density gradient centrifugation with washing from PGI2-supplemented platelet-rich plasma (PRP). We have assessed the degree of erythrocyte and leukocyte contamination, recovery of platelets, morphological features, activation status, and reactivity of isolated platelets. Using our protocol, we were able to get a preparation free from contaminations, representing well the platelet population prior to the isolation in terms of size and activity. Besides this, we have obtained approximately 2 times more platelets from the same volume of blood compared to the most widely used method. From 10 ml of whole citrated blood we were able to get on average 2.7 mg of platelet-derived protein. The method of platelet isolation presented in this paper can be successfully applied to tests requiring very pure platelets, reflecting the circulating platelet state, from a small volume of blood.

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.

Clinical efficacy of a combination of Percoll continuous density gradient and swim-up techniques for semen processing in HIV-1 serodiscordant couples.

To evaluate the clinical efficacy of a procedure comprising a combination of Percoll continuous density gradient and modified swim-up techniques for the removal of human immunodeficiency virus type 1 (HIV-1) from the semen of HIV-1 infected males, a total of 129 couples with an HIV-1 positive male partner and an HIV-1 negative female partner (serodiscordant couples) who were treated at Keio University Hospital between January 2002 and April 2012 were examined. A total of 183 ejaculates from 129 HIV-1 infected males were processed. After swim-up, we successfully collected motile sperms at a recovery rate as high as 100.0% in cases of normozoospermia (126/126 ejaculates), oligozoospermia (6/6), and asthenozoospermia (36/36). The recovery rate of oligoasthenozoospermia was 86.7% (13/15). In processed semen only four ejaculates (4/181:2.2%) showed viral nucleotide sequences consistent with those in the blood of the infected males. After using these sperms, no horizontal infections of the female patients and no vertical infections of the newborns were observed. Furthermore, no obvious adverse effects were observed in the offspring. This protocol allowed us to collect HIV-1 negative motile sperms at a high rate, even in male factor cases. We concluded that our protocol is clinically effective both for decreasing HIV-1 infections and for yielding a healthy child.

Transgressive, reiterative selection by continuous buoyant density gradient centrifugation of Dunaliella salina results in enhanced lipid and starch content

Microalgae can serve as useful feedstocks for biofuel production as they can be grown with fresh, brackish, or salt water and their lipid and starch contents can be manipulated to create customized feedstocks for different classes of biofuels. Continuous buoyant density gradient centrifugation (CBDGC) was used to perform reiterative, transgressive selection to isolate wildtype and ethyl methanesulfonate-mutagenized Dunaliella salina cells with enhanced lipid and starch production. Sixty rounds of transgressive selection resulted in the isolation of cell populations with significantly lower or higher buoyant densities. Lipid content in the low-density populations was enhanced by 1.2- to 2.9-fold in wildtype cells and 1.3- to 2.3-fold in mutagenized cells as measured by Nile Red dye staining, but the lipid content differences were not significant when quantified by liquid chromatography–tandem mass spectroscopy possibly due to the composition of the lipid pools measured by these contrasting techniques. In contrast, starch content in the high-density populations was increased by 2-fold in wild type cells and 1.4- to 1.6-fold in mutagenized cells, respectively. The observed alterations in lipid and starch contents appeared to be stable after more than 70weeks (392 cell generations). CBDGC-based selection provides a useful and accessible technological alternative to genetic engineering approaches for the customization of microbial biofuel feedstocks.

CENTRIFUGAÇÃO DE ESPERMATOZOIDES OVINOS EM GRADIENTE DE DENSIDADE CONTÍNUO: EFEITO SOBRE A TAXA DE PRENHEZ E PROPORÇÃO DO SEXO

Este trabalho teve como objetivo avaliar a eficiência de gradientes de densidade contínuos de Percoll e OptiPrep para a separação de espermatozoides portadores do cromossomo X, e avaliar a influência sobre a viabilidade espermática, taxa de prenhez e proporção do sexo. Para isso, 100 milhões de espermatozoides foram depositados em cada tubo contendo o gradiente de densidade, previamente preparados, em tubos de poliestireno, 24 horas antes da centrifugação e mantidos a 4 °C. Centrifugou-se a 500 x g por 15 minutos a 22 °C e, posteriormente, os sobrenadantes foram aspirados e os espermatozoides recuperados no fundo dos tubos. Uma amostra foi avaliada antes a após a centrifugação para verificação da motilidade e vigor espermático. Os espermatozoides foram utilizados para a inseminação artificial em tempo fixo. Os resultados indicaram que a centrifugação não afetou a viabilidade espermática e a taxa de prenhez, entretanto não houve desvio da proporção do sexo para fêmeas. Mais estudos são necessários para determinar a necessidade de adaptação da técnica de centrifugação para a espécie ovina e assim obter o desvio da proporção do sexo como na espécie bovina.

Differences in uptake and killing of pathogenic and non-pathogenic bacteria by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone).

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non-pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi-granulocytes) have the main function in phagocytosis of both pathogenic and non-pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non-virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post-inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non-virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.

Evidence for the functioning of a Cl−/H+ antiporter in the membranes isolated from root cells of the halophyte Suaeda altissima and enriched with Golgi membranes

Cl−/H+ exchange activity in the membranes isolated from the root cells of the halophyte Suaeda altissima (L.) Pall. was originally revealed and characterized. The membrane vesicles were isolated by centrifugation of microsomes in a continuous iodixanol density gradient. The highest activity of latent inosine phosphatase, a marker of Golgi membranes, was localized in the upper part of the gradient, indicating its enrichment with Golgi membranes. The same part of the gradient was characterized by the highest Cl−/H+ exchange rate. The Cl−/H+ exchange activity was detected as electrogenic ΔpCl-dependent H+ transport monitored as changes in differential absorbance of a ΔpH-probe acridine orange, or as changes in fluorescence excitation spectrum of a pH-probe pyranine loaded into the vesicles. Generation of transmembrane electric potential (Δψ) during the Cl−/H+ exchange was assayed as changes in differential absorbance of a Δψ-probe safranin O. Establishing the transmembrane ΔpCl inward vesicles resulted in H+ efflux sensitive to DIDS (4,4′-diisothiocyano-2,2′-stylbene-disulfonic acid), an inhibitor of chloride transporters and channels, and generation of Δψ negative inside. To maintain the ΔpCl-dependent H+ efflux from the vesicles, either the presence of a penetrating cation tetraphenylphosphonium neutralizing negative charges inside the vesicles or null K+ diffusion potential across the membranes was required. The results demonstrate the activity of an electrogenic Cl−/H+ antiporter in the fraction enriched with Golgi membranes. We hypothesize that the Cl−/H+ antiporter is involved into the regulation of cytoplasmic Cl− concentrations by vesicular trafficking of Cl− from the cytoplasm to the vacuole by endosomes, derivatives of Golgi membranes.

Tests of FARICH prototype with precise photon position detection

In June 2012 a FARICH prototype from Philips Digital Photon Counting (PDPC) based on a photon camera with dimensions of 200×200mm has been tested at CERN. Remarkable particle separation has been achieved with a 4-layer aerogel sample: the π/K separation at a 6GeV/c momentum is 3.5σ, the μ/π separation is 5.3σ at 1GeV/c. The analysis of the data has shown that the main contribution to the accuracy of the ring radius measurement comes from aerogel. The development of focusing aerogels is proceeding in two main directions: tuning of production technology of multilayer blocks and development of a new production method with continuous density (refractive index) gradient along the block depth. The beam test was carried out in December 2012–January 2013 at the electron beam test facility at the VEPP-4M e+e− collider. The goal of this test was to measure different single layer and focusing aerogel samples, both multilayer and gradient. Aerogel samples were tested with a PDPC FARICH prototype. A part of DPC SPADs in each pixel was disabled to form an active area of 1×1mm2. The collected data proved that gradient aerogel samples focus Cherenkov light.

Aerogel for FARICH detector

We present our current experience in preparation of focusing aerogels for the Focusing Aerogel RICH detector. Multilayer focusing aerogel tiles have been produced in Novosibirsk by a collaboration of the Budker Institute of Nuclear Physics and Boreskov Institute of Catalysis since 2004. We have obtained 2–3–4-layer blocks with the thickness of 30–45mm. In 2012, the first samples of focusing blocks with continuous density (refractive index) gradient along thickness were produced. This technology can significantly reduce the contribution from the geometric factor of the radiator thickness to the resolution of the measured Cherenkov angle in the FARICH detector. The special installation was used for automatic control of reagents ratio during the synthesis process. The first samples were tested using the digital radiography method and on the electron beam with the FARICH prototype.

Mature VLDL triggers the biogenesis of a distinct vesicle from the trans-Golgi network for its export to the plasma membrane

Post-Golgi trafficking of mature VLDL (very-low-density lipoprotein) is crucial in maintaining normal TAG (triacylglycerol) homoeostasis of hepatocytes; however, the mechanism that regulates the exit of mature VLDL from the TGN (trans-Golgi network) is not known. We developed an in vitro TGN-budding assay that allowed us to examine the formation of secretory vesicles from the TGN in primary rat hepatocytes. We isolated TAG-rich PG-VTVs (post-TGN VLDL transport vesicles) using a continuous sucrose density gradient. PG-VTVs were distributed in low-density fractions, whereas protein transport vesicles were present in relatively higher-density fractions of the same sucrose gradient. EM revealed large intact PG-VTVs ranging 300-350 nm in size. The biogenesis of PG-VTVs from the TGN required cytosol, ATP, GTP hydrolysis and incubation at 37°C. PG-VTVs concentrated the VLDL proteins: apolipoproteins apoB100, apoAIV, apoAI and apoE, but did not contain either albumin or transferrin. Proteinase K treatment did not degrade VLDL core proteins, suggesting that PG-VTVs were sealed. PG-VTVs were able to fuse with and deliver VLDL to the PM (plasma membrane) in a vectorial manner. We conclude that we have identified a new TGN-derived vesicle, the PG-VTV, which specifically transports mature VLDL from the TGN to the PM.

An effective purification method using large bottles for human pancreatic islet isolation

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.

Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication

An increasing number of studies demonstrate that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, is involved in the infection processes of a variety of pathogens. It can be hijacked by various viruses to facilitate their replication. In this study, we found that PRRSV infection significantly increases the number of double- or single-membrane vesicles in the cytoplasm of host cells in ultrastructural analysis. Our results showed the LC3-I was converted into LC3-II after virus infection, suggesting the autophagy machinery was activated. We further used pharmacological agents and shRNAs to confirm that autophagy promoted the replication of PRRSV in host cells. Confocal microscopy analysis showed that PRRSV inhibited the fusion between autophagosomes and lysosomes, suggesting that PRRSV induced incomplete autophagy. This suppression caused the accumulation of autophagosomes which may serve as replication site to enhance PRRSV replication. It has been shown that NSP2 and NSP3 of arterivirus are two components of virus replication complex. We also found in our studies that NSP2 colocalized with LC3 in MARC-145 cells by performing confocal microscopy analysis and continuous density gradient centrifugation. Our studies presented here indicated that autophagy was activated during PRRSV infection and enhanced PRRSV replication in host cells by preventing autophagosome and lysosome fusion.

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Size and Shape Separation of Ag Nanoparticles by a Continuous Density Gradient Centrifugation

In order to get highly monodispersed nanoparticles, a post-synthesis separation of nanopaticles polydisersed in size and shape is often desired, although the experimental conditions for the synthesis of nanoparticles are carefully controlled. Using continuous density gradient centrifugation, Ag nanospheres, nanocubes, and nanowires can be separated from polydisperse samples. Size and dimension analysis by sanning electron microscopy and absorption spectroscopy prove that density gradient centrifugation is a powerful method to obtain monodisersed nanoparticles.

An apparatus to control and automate the formation of continuous density gradients

A common problem associated with making a continuous density gradient is the unwanted mixing of liquids as the gradient solution is being delivered from the gradient mixer to the centrifuge tube. We show that by using a Styrofoam float on a thin drawn-out glass pipette, liquids from the gradient mixer will flow down the outside surface of the glass fiber and contact the float, after which the liquid spreads across the gradient surface without unwanted mixing. As the liquid in the tube rises, the float rises up the glass fiber, thereby allowing the creation of gradients without the need to monitor the process.

Evidence of Endoplasmic Reticulum Stress Mediating Cell Death in Transplanted Human Islets

A key limitation to the success of islet transplantation is islet cell exhaustion and cell death during islet isolation and following transplantation. Endoplasmic reticulum (ER) stress has been identified as an important mechanism in the development of β-cell dysfunction, cell death, and diabetes. This study investigated the role of ER stress in islet loss during human islet isolation and posttransplantation in a diabetic athymic mouse model. Islets were isolated from human organ donor pancreata using intraductal enzymatic dissociation and continuous density gradient purification. ER stress mediators were assessed by Western blot and by RT-PCR. Caspase-3 activity was quantified by a bioluminescent peptide cleavage assay. Normal and streptozotocin-treated diabetic nude mice were transplanted with 2,000 IEQ of human islets under the kidney capsule and the grafts were harvested 3 or 28 days after transplantation. The grafts were analyzed for the presence for ER stress signals by immunohistochemistry. Isolated islets demonstrated higher levels of ER chaperone Bip, ER stress mediators eIF2α, ATF, spliced XBP-1, and CHOP, and also ER stress-associated apoptotic signals like JNK, caspase-3/7, and cleaved PARP. Donor pancreatic tissue did not show expression of any of these ER stress mediators. After transplantation, low expression of only protective ER stress mediators was evident in the grafts from the normal recipients. In contrast, both protective and apoptotic ER stress mediators were highly expressed in the grafts of hyperglycemic mice. ER stress mediators were induced during islet isolation and may contribute to islet apoptosis and cell death. Islet isolation activates ER stress and apoptotic pathways in isolated islets. Hyperglycemia may prolong this ER stress signal in engrafted islets, converting the protective aspects of the ER stress response to a proapoptotic response and thus contribute to deterioration of β-cell function and survival.

Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin‐1

N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line. BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot. BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB(1) receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change. Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.