Percoll Centrifugation Research Articles

Elevated mtDNA content in RBCs promotes oxidative stress may be responsible for faster senescence in men

BackgroundBoth we and others have found that RBC counts are significantly lower in older compared to younger. However, when gender is factored in, a significant age-related decrease of RBC counts is observed only in men but not in women. MethodsqPCR and confocal microscopy were used to detect the presence of mtDNA in RBCs. Flow cytometry and specific inhibitors were used to determine how RBCs uptake cf-mtDNA. The peripheral blood was collected from 202 young adults and 207 older adults and RBC and plasma were isolated. The levels of TLR9+RBCs and apoptotic RBCs after uptake of cf-mtDNA by RBCs were measured by flow cytometry. The kit detects changes in SOD and MDA levels after cf-mtDNA uptake by RBCs. Young RBCs (YR) and old RBCs (OR) from single individuals were separated by Percoll centrifugation. ResultsWe found a significant decrease in RBC counts and a significant increase in the RDW with aging only in men. We also found that significantly elevated mtDNA content in RBCs was observed only in men during aging and was not found in women. Further studies demonstrated that RBCs could take up cf-mtDNA via TLR9, and the uptake of mtDNA might lead to a decrease in the RBC number and an increase in RDW due to an increase of oxidative stress. ConclusionsThe RBC mtDNA content might be a potential marker of RBC aging and the elevated RBC mtDNA content might be the cause of faster senescence in males than females.

Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris.

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris-an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research-using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1β, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1β and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

Myeloid-derived Suppressor Cells Activate Liver Natural Killer Cells in a Murine Model in Uveal Melanoma.

Elevated myeloid-derived suppressor cells (MDSCs) in many malignancies are associated with the increased risk for metastases and poor prognosis. Therefore, a mouse model of intraocular melanoma was established to explore how MDSCs influence liver metastases. In this study, murine B16LS melanoma cells were transplanted into the posterior compartment (PC) of the eye of C57BL/6 mice. Leucocytes from the liver of naive mice and mice bearing melanoma liver metastasis were isolated using isotonic Percoll centrifugation, examined by flow cytometry for their expression of Gr1, CD11b, F4/80, RAE-1, and Mult-1, and further isolated for MDSCs and natural killer (NK) cells. The effects of MDSCs on NK cells were tested by coculturing and assessing the ability of NK cells to produce interferon-gamma (IFN-γ) by ELISA and NK cell cytotoxicity by 3H-thymidine incorporation assay. The impact of IFN-γ on liver metastases was examined via selectively depleting IFN-γ in vivo. The results showed that mice with liver metastases had increased levels of CD11b+Gr1+F4/80+ as well as CD11b+Gr1+F4/80- MDSCs. MDSCs significantly enhanced the generation of IFN-γ together with the cytotoxicity of the NK cells. Furthermore, these effects were cell-cell contact-dependent. Although IFN-γ was not of a toxic nature to the melanoma cells, it profoundly inhibited B16LS cell proliferation. Depleting IFN-γ in vivo led to increased liver metastases. All these findings first revealed that MDSCs accumulated in liver metastasis of intraocular melanoma could activate the NK cells to produce an effective anti-tumor immune response. Thus, the MDSCs' performance in different tumor models would need more investigation to boost current immunotherapy modalities.

P-061 What is the optimal number of IUIH cycles before changing treatment ?

Abstract Study question Is there an optimal number of intrauterine inseminations before offering another treatment for unexplained infertility, taking into account cumulative pregnancy rate and drop out? Summary answer Up to 3-4 intrauterine inseminations are advisable for unexplained and mild male infertility, as this will provide the optimal chances for the patient. What is known already Intrauterine insemination has been used as a first line treatment for unexplained infertility and mild male subfertility. However, during the years great debate and only few randomised controlled studies has been published supporting this. It has been shown that up to 3 inseminations are comparable to an IVF cycle. Further, multiple gestations can be managed and reduced in inseminations as well. Thus, up to 3 cycles of intrauterine inseminations can increase the chances of a healthy offspring and in controlled trials are comparable to an IVF cycle. Study design, size, duration The study is a retrospective cohort study including all patients in treatment for unexplained infertility, anovulation disorders or mild male factor from 2005 to 2022 in a single fertility clinic. Inclusion criteria: more than 1 year of infertility, normal or moderate sperm parameters. A total of 3729 women were included and all followed until pregnancy or drop out. A total of 9459 cycles were included. Participants/materials, setting, methods Women between 18 and 40 years were included. All had more than 1 year of infertility and suffered from unexplained infertility, mild male factor or ovulation disorders. All women received a mild oral tamoxifen or clomiphene stimulation for 5 days and when the leading follicle was 17 mm in diameter an ovulation induction with Fyrmadel 0,25 mg was used. All inseminations after Percoll centrifugation. Pregnancy in 12 weeks were recorded including number of gestations. Main results and the role of chance We found a cumulative pregnancy rate for 3 cycles to be 29%, for 4 cycles 32%, for 5 cycles 35%, for 6 cycles 37%, and a corresponding cumulative drop out of; for 3 cycles; 33%, for 4 cycles;43% for 5 cycles; 50%, for 6 cycles 55%. Multiple gestations were found to be 4%. Using this large data material, we found that 3-4 intrauterine inseminations were acceptable due to the cumulative pregnancy rate and drop numbers. Increasing the number to more cycles did not benefit the patient’s chance significantly and should not be recommended. The study was able to identify all cycles from any individual person starting the treatment and therefore the drop out form a main fertility clinic could be estimated properly. No hyperstimulation’s syndrome were identified and a multiple gestational risk of 4 % were identified. Limitations, reasons for caution A retrospective study from 2005 to 2021 with the limitations that a retrospective carries. The lab procedure/stimulation protocol were the same which makes the results robust. This study involved a large number of patients (3729) every cycle were followed until they got pregnant and dropped out until the 6th cycle. Wider implications of the findings Intrauterine insemination is a safe and efficient treatment option for a group of infertile women. This study describes the relative high drops out after 4-5 cycles and a cumulative pregnancy rate of 30-33% following 3-4 cycles. hereafter the cumulative pregnancy rate does not rise significantly and drop out increases. Trial registration number na

Sorting and cryopreservation of goat sperm with or without phenolic compounds

ABSTRACT The objectives of this study were to evaluate goat sperm sorting in continuous Percoll® density gradients and gamete freezability, in the presence or absence of phenolic antioxidants. For this, semen pools were sorted, frozen, and evaluated. The non-selected group (NSg) presented lower progressive motility (PM), linearity (LIN), straightness (STR), and wobble (WOB) than the selected groups, and straight line velocity (VSL) compared to those with catechin or resveratrol. The amplitude of lateral head displacement (ALH) was higher in NSg, and quercetin reduced the mitochondrial membrane potential (MMP). After thawing, the NSg presented lower PM than the selected groups, VSL and VAP (average path velocity) than the selected group with or without catechin, LIN and WOB than the selected with or without catechin or resveratrol, and STR than the selected with catechin. Moreover, NSg presented higher ALH and BCF than the samples selected with or without catechin. Plasma membrane integrity and intact and living cells were higher in the selected groups, and MMP was lower in the NSg and the selected group with quercetin. Thus, centrifugation in Percoll® continuous density gradients is a viable methodology to select goat sperm compatible with the freezing, especially in the presence of catechin or resveratrol.

Chlorogenic acid improves the quality of boar semen processed in Percoll

This study aimed to evaluate if the addition of chlorogenic acid (ChA) to semen extenders improves the quality of cooled boar semen processed in Percoll. The experimental design was randomized blocks (ejaculates) in a 2×3 factorial (with or without Percoll, and three antioxidant systems: a negative control, without supplementation, a positive control – vitamin E, and ChA), totaling six treatments and 12 repetitions. ChA and vitamin E (VE) were added at 4.5 mg/ml and 400 μg/ml in extender, respectively. At 0, 48 and 72h of storage at 15ºC, 80 ml insemination doses each containing 2.0 billion sperm cells were submitted to centrifugation in Percoll. The use of Percoll impaired (P<0.01) all motility patterns but decreased (P<0.01) the number of abnormal cells at 0, 48 and 72h of storage. Both VE and ChA improved (P<0.05) the total motility after Percoll processing, but only in semen stored for 48h. The same effect was not observed (P>0.05) in semen stored for 72h. ChA improved (P<0.05) the total motility of the semen stored for 72h, but this effect was not observed (P>0.05) when the semen was processed in Percoll. The antioxidants had no effect (P>0.05) on the viability and integrity of the acrosome, but ChA reduced (P<0.05) the number of abnormal cells at 0h, while VE increased the number of abnormal cells in semen stored for 72h, independent of the use of Percoll. There was no effect (P>0.05) of antioxidants or Percoll on the concentration of malondialdehyde in seminal plasma. The use of Percoll had no effect (P>0.05) on the cholesterol efflux, but ChA increased (P<0.05) this parameter at 0h and reduced (P<0.05) in the semen stored for 72h not processed with Percoll. In conclusion, the addition of ChA to semen extenders improved the quality of boar semen processed or not in Percoll.

112 The effect of biological extenders on invitro induction of the acrosome reaction in bovine spermatozoa

Biological extenders in semen are used to protect the integrity of the sperm cells during cryopreservation. The two most common extenders are egg yolk and milk, and initial experiments describing heparin capacitation for IVF were conducted with semen frozen in egg yolk extender. However, semen of many bulls used for IVF is frozen in milk-based extender, and it is not clear whether this affects the response to heparin capacitation. This study aims to assess whether the use of milk- or egg-based extenders in frozen-thawed bovine semen affects sperm's ability to undergo the acrosome reaction, utilising either the endogenous progesterone pathway or calcium ionophore A23187 to induce the acrosome reaction following heparin capacitation. Frozen semen from 12 bulls (6 using egg yolk citrate-based extender containing 20% egg yolk, and 6 using milk-based extender) was used for no less than 3 replicates each. All semen was commercially processed and cryopreserved using the same methodology. Semen was thawed and separated using a Percoll gradient and centrifugation at 400×g for 10min, then incubated for 4h in a capacitating medium containing heparin (10μgmL−1) at a concentration of 1×106 spermmL−1. Capacitated sperm were then introduced to progesterone (10μM) or A23187 (10 μM) for acrosome induction and stained with fluorescein isothiocyanate-conjugated peanut agglutinin (5μgmL−1). After treatment and staining, the sperm were analysed using flow cytometry to determine the percentage of populations with fluorescent acrosomes, indicating an exposed acrosome and an ongoing acrosome reaction. Fluorescent microscopy was also used to confirm fluorescence in acrosome-reacting samples. Results were analysed as a 2×3 factorial design by ANOVA with extender and acrosome induction treatment as factors. For egg yolk-extended semen, the mean percent of acrosome-reacted sperm was 83.7 (±6.8), 81.8 (±7), and 15.0 (±7.5) for sperm treated with progesterone, A23187, and heparin only, respectively. Compared to milk-extended semen, for which the mean percent acrosome-reacted was 15.0 (±8.2), 14.8 (±7.4), and 12.0 (±6.8) for progesterone, A23187, and heparin only, respectively. Significant differences were observed between extenders, with egg yolk-extended semen having a significantly greater response to acrosome reaction-inducing agents than semen extended with milk-based extenders (P&amp;lt;0.05). Also, it was observed that progesterone induced a similar percentage of acrosome reactions as A23187. Variation between bulls in the response to heparin capacitation has been observed and cannot be eliminated in this study. The difference observed between extenders warrants further study with a split ejaculate design. The possibility that the semen extender used during cryopreservation affects the response to heparin capacitation suggests that this should be accounted for in IVF protocols to increase the efficiency of this technology.

Impact of Percoll purification on isolation of primary human hepatocytes

Research and therapeutic applications create a high demand for primary human hepatocytes. The limiting factor for their utilization is the availability of metabolically active hepatocytes in large quantities. Centrifugation through Percoll, which is commonly performed during hepatocyte isolation, has so far not been systematically evaluated in the scientific literature. 27 hepatocyte isolations were performed using a two-step perfusion technique on tissue obtained from partial liver resections. Cells were seeded with or without having undergone the centrifugation step through 25% Percoll. Cell yield, function, purity, viability and rate of bacterial contamination were assessed over a period of 6 days. Viable yield without Percoll purification was 42.4 × 106 (SEM ± 4.6 × 106) cells/g tissue. An average of 59% of cells were recovered after Percoll treatment. There were neither significant differences in the functional performance of cells, nor regarding presence of non-parenchymal liver cells. In five cases with initial viability of <80%, viability was significantly increased by Percoll purification (71.6 to 87.7%, p = 0.03). Considering our data and the massive cell loss due to Percoll purification, we suggest that this step can be omitted if the initial viability is high, whereas low viabilities can be improved by Percoll centrifugation.

Differences in mitochondrial Ca2+ handling during challenges of CaCl2 pulses in brain synaptic and non‐synaptic mitochondria: implications for differential Ca2+ buffering

Mitochondria play a key role in cytosolic Ca2+ regulation and buffering, with Ca2+ uptake mainly via the inner membrane mitochondrial Ca2+ uniporter (MCU), and efflux of mitochondrial Ca2+ largely via the Na+/Ca2+ exchanger (mNCE). Previous reports have shown brain synaptic and non‐synaptic mitochondria display marked differences in Ca2+ uptake before permeabilization of the mitochondrial membranes, the so‐called permeable transition pore (mPTP). However, the kinetics of mitochondrial Ca2+ handling and its implications for Ca2+ buffering after boluses of CaCl2 have not been reported. In this study, we aimed to define the kinetics of Ca2+ handling in synaptic and non‐synaptic mitochondria isolated from rat brains via discontinuous percoll centrifugation followed by differential centrifugation. Respiratory control index (RCI), and subsequent changes in calcium retention capacity (CRC) and membrane potential (ΔΨm), were assessed in isolated mitochondria. CRC and ΔΨm were evaluated with Fura 4F penta‐K salt and TMRM dyes, respectively, using fluorescence spectrophotometry (Photon Technology). Mitochondria were energized with glutamate and malate, and CRC and ΔΨm were assessed during state 2 respiration. To investigate the potential role of the mNCE in Ca2+ handling, the NCE blocker CGP37157 (CGP) was used. In addition, experiments were conducted to determine the expression levels of the MCU and mNCE in the two mitochondria pools using western blot. Mitochondrial RCI was higher in the synaptic group compared to the non‐synaptic group, which is consistent with past results. Ca2+ uptake and retention without CGP were lower in synaptic mitochondria compared to non‐synaptic mitochondria. Addition of CGP markedly enhanced CRC in the synaptic mitochondria to levels found in non‐synaptic mitochondria without CGP. In contrast, the CGP induced changes in CRC were not observed in the non‐synaptic mitochondria. Membrane potential depolarization occurred in both synaptic and non‐synaptic mitochondria as matrix free Ca2+ accumulated, reflecting the decrease in Ca2+ sequestration or Ca2+ efflux in the non‐synaptic and synaptic mitochondria, respectively. MCU expression was higher in the non‐synaptic mitochondria compared to the synaptic mitochondria, whereas mNCE expression was not different. The increased Ca2+ uptake and retention in the presence of CGP in the synaptic mitochondria implies that mNCE is active in synaptic mitochondria, which leads to faster extrusion of added Ca2+ before it is sequestered. The lack of change in Ca2+ kinetics in non‐synaptic mitochondria suggests that NCE activity is lower. The ΔΨm and CRC results likely indicate that synaptic mitochondria can buffer Ca2+ like that in non‐synaptic mitochondria, but only do so when the mNCE is blocked. We propose that synaptic mitochondria may have increased mNCE activity to regulate its matrix free Ca2+, while non‐synaptic mitochondria depend mostly on buffering of the free Ca2+; the implications for these differential responses may be related to the physiological role of these two populations of mitochondria.Support or Funding InformationFunding: NIH T35 HL072483 and MCW‐AHWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Variances in cellular sedimentation behavior as an effective enrichment method of hydrocarbon-overproducing Micrococcus luteus strains

BackgroundAliphatic hydrocarbons of microbial origin are highly interesting candidate biofuels because these molecules are identical or very similar to the main components of petroleum-based gasoline and diesel fuels. The high-GC Gram-positive bacterium Micrococcus luteus is capable of naturally synthesizing long-chain, iso- and anteiso-branched alkenes which are formed via the head-to-head condensation of fatty acid thioesters by a dedicated enzyme system. The present study describes the relation we observed between olefin production and cell buoyancy in Micrococcus luteus and the use of this phenotype to simply and efficiently separate cells from a mixture based on their hydrocarbon content.MethodsWe generated M. luteus mutants producing different amounts of olefins and used them in mixing and sedimentation experiments, olefin content analysis by GC-MS and in equilibrium centrifugation in Percoll gradients.ResultsWe found well-detectable differences in the buoyant densities of the examined strains, which correlated with the amounts of hydrocarbons produced by the cells. We also demonstrate how our observations can be used to simply and efficiently fractionate cells based on their hydrocarbon content.ConclusionsIn summary, we show that cultures of M. luteus cells sediment at distinct rates depending on the amounts of alkenes produced. Our results indicate that buoyant cell density is the primary cause for the observed differences in sedimentation behaviour. The simple separation strategy described here can be a valuable tool in various mutagenesis and enrichment protocols, aimed at generating and isolating strains with increased olefin productivity.

Donor sperm production in heterologous recipients by testis germ cell transplantation in the dromedary camel.

The object of this study was to investigate if testis germ cell transplantation (TGCT) into a heterologous recipient would result in donor-origin spermatogenesis in the dromedary camel. First, we investigated a workable protocol for TGCT in camels, including donor cell isolation, enrichment by density gradient centrifugation (Percoll and Bovicoll), rete testis injection and microsatellite detection of donor and recipient genotypes. Second, the effects of three doses of Dolichos biflorus agglutinin (DBA), a glycoprotein that specifically binds to gonocytes or Type A spermatogonia, on testis germ cell depletion were investigated by direct injection into the rete testis of a male camel. Seven recipients were prepared with DBA treatment, two males were castrated at 4 weeks for depletion assessment and the remaining five received donor cells 4-6 weeks after treatment. On average, ~17 million cells were isolated per gram of testis tissue, with 19.5±1.9% DBA-positive (DBA+) cells. Percoll centrifugation yielded a 1.5-fold increase in DBA+ cells while Bovicoll centrifugation produced a 2.5-fold increase from the input cells of 18.6±2.1% DBA+ cells. Semen was collected from the recipients 13-20 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In two of the five recipients, all semen samples were shown to be positive for donor-derived cells. These results demonstrate for the first time that: (1) heterologous testicular germ cell transplantation in camels is feasible and the recipients are able to produce spermatozoa of donor origin and (2) DBA can be used effectively to deplete endogenous stem cells.

The expression characteristics of FAM71D and its association with sperm motility.

What are the features of FAM71D (Family with sequence similarity 71, member D) expression and is there an association between FAM71D expression and sperm motility? FAM71D, a novel protein exclusively expressed in the testis, is located in sperm flagella and is functionally involved in sperm motility. Some testis-specific proteins have been reported as potential diagnostic biomarkers to evaluate the spermatogenesis process and sperm quality. We have identified a novel testis-specific protein, FAM71D, through microarray data analysis, yet little is known about its expression and function. FAM71D mRNA and protein expression was quantified during mouse testis development. Its localization in germ cells was detected by dual-labeled immunostaining in testis sections and sperm smears. The clinical significance was assessed by comparing FAM71D expression in spermatozoa from normozoospermic controls and asthenozoospermic patients. Testes were dissected from C57BL/6 J male mice at postnatal ages of 1, 2, 3, 4, 6, 8 weeks and 6 months, and sperm was collected from cauda epididymides of adult mice by the swim-up method. Human spermatozoa were isolated from 100 human semen samples by density gradient Percoll centrifugation. RT-qPCR and western blot were performed to semi-quantify the expression of FAM71D in mouse testis, and in the ejaculated spermatozoa of normozoospermic controls and asthenozoospermic patients. Immunofluorescence staining was used to detect the localization of FAM71D. Co-immunoprecipitation assay was performed to evaluate the interaction between FAM71D and calmodulin. An antibody blocking assay was employed to assess the role of FAM71D in sperm motility. Our results showed that FAM71D was exclusively expressed in the testis in an age-dependent manner. FAM71D expression exhibited dynamic change in the cytoplasm of spermatids during spermiogenesis and was finally retained in sperm flagella. FAM71D could interact with calmodulin. Use of anti-FAM71D antibody on sperm significantly decreased sperm motility. Expression level of FAM71D was markedly reduced in the ejaculated spermataozoa of asthenozoospermic patients (P < 0.05), and this was correlated with sperm progressive motility (r = 0.7435, P < 0.0001). N/A. The sample size was limited and it is necessary to verify the correlation of FAM71D expression with sperm motility in larger cohorts. Furthermore, our results were descriptive and follow-up studies would be needed to elucidate the detailed role of FAM71D in sperm motility. This is the first systematic study to document the expression of endogenous FAM71D and a function for FAM71D in sperm motility. It provides new insights into our understanding of sperm motility regulation and causes of male infertility. This study was funded by the National Natural Science Foundation of China, Guangdong Natural Science Foundation and the Shenzhen Project of Science and Technology. The authors have no competing interests.

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed. Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficoll and Spontaneous Sedimentation Methods

ABSTRACTStudies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.

Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System

An understanding of how to safely apply intraoperative blood salvage (IBS) in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2), gastrocarcinoma (SGC7901), and colonic carcinoma (SW620) tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy), tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery.

Obtaining chicken primordial germ cells used for gene transfer: in vitro and in vivo results.

Recently, several attempts have been made to create a generation of transgenic chickens via chimeric intermediates produced by primordial germ cells (PGCs) transfer. This study aimed to compare the influences of different chicken PGCs isolated from circulating blood (bPGCs) or gonads (gPGCs), purification (ACK, Percoll or trypsin) and transfection methods (electroporation or lipofection) on the expression of transgenes in vitro and the migration of modified donor cells to the recipient gonads. The highest average frequency of pEGFP-N1 plasmid-transfected bPGCs (75.8 %) was achieved with Percoll density gradient centrifugation and electroporation. After ammonium chloride-potassium (ACK) treatment and lipofection, in vitro transgene expression was only detected in 35.2 % of bPGCs. Chimeric chickens were produced from these purified, transfected and cultured cells, and the transgene was detected in the gonads of 44 and 42 % of the recipient embryos that had been injected with bPGCs and gPGCs, respectively. These data confirmed that the combination of PGC purification via Percoll centrifugation and electroporation was an effective method for producing transgenic chickens. Subsequently, we used this method with expression vectors for gene hIFNα 2a/hepatitis B virus surface antigen (HBsAg) under the control of the ovalbumin promoter to generate G0 transgenic chickens. Consequently, we observed that 4.9 % of the hens and 3.5 % of the roosters carried the hIFNα 2a gene, whereas 16.7 % of the hens and 2.4 % of the roosters carried the HBsAg gene, thus undisputedly confirming the exceptional effectiveness of the applied methods.

In vitro screening method for the evaluation of histamine release from rat peritoneal cells

Rat peritoneal mast cells offer a convenient approach for the evaluation of histamine release by chemicals. Peritoneal mast cells obtained by centrifugation on density gradients are routinely used for the assessment of histamine release. Manipulation of the cells during this purification step may lead to cell membrane damage and spontaneous release of histamine. The purpose of the present study was to evaluate two procedures of peritoneal cell purification. Rat peritoneal cells were purified (i) by three centrifugations in PIPES only (PIPES purification) or (ii) by three centrifugations in PIPES followed by density gradient centrifugation in Percoll (PIPES/Percoll purification). The effect of a reference compound 48/80 was evaluated at two or three concentrations (0.1, 1 and 10 μg/ml) under several experimental conditions

Insight into the overconsumption of ammonium by anammox consortia under anaerobic conditions.

This study gives a systematic profile of ammonium overconsumption in anammox consortia beyond the stoichiometry ratio, with aim to reveal another ammonium removal process by oxidants OH· without nitrite under anaerobic conditions. Percoll centrifugation, flow cytometry, cloning and chemical analysis demonstrated that this ammonium removal was related to low Planctomycetes and high biomass decay. Armatimonadetes, Acidobacteria, Verrucomicrobia and Nitrosomonas initially existed in the consortia with the overconsumption of ammonium, and their levels then declined with decreasing overconsumption. The inevitable release of oxidants, such as OH, during bacterial inhibition or decay was found to be a potential source of electron acceptors capable of oxidizing ammonium. Anaerobic ammonium removal without nitrite could occur in anammox consortia. However, it was unsteady and was not sustained over a long period. Under suitable conditions, anammox bacteria competed with others and became the dominant micro-organism responsible for the simultaneous removal of ammonium and nitrite. This investigation will have importance to better understand the anammox process operation. The overconsumption of ammonium could be used as an indicator for fluctuations in anammox cultivation.

A pan caspase inhibitor decreases caspase-1, IL-1α and IL-1β, and protects against necrosis of cisplatin-treated freshly isolated proximal tubules

Caspase-1, IL-1α, and IL-1β are known to be activated in the NLRP3 inflammasome. The inflammasome is activated mostly in inflammatory cells. The presence of inflammasome proteins in proximal tubules (PTs) and the effect of cisplatin-treatment or caspase inhibition on inflammasome proteins in PTs are not known. The aim of this study was to investigate the effect of cisplatin on inflammasome proteins in freshly isolated PTs and also to determine the effect of caspase inhibition on inflammasome proteins and PT injury. PTs were isolated using collagenase digestion and Percoll centrifugation. After recovery period, freshly isolated PTs were incubated with vehicle, 50 µM cisplatin or 50 µM cisplatin plus 50 µM pan caspase inhibitor, QVD-OPH. PTs treated with 50 µM cisplatin showed Propidium Iodide staining indicative of necrosis. Necrotic cells (%) were 2.2 in Vehicle-treated, 37.7 in Cisplatin-treated (p < 0.05 vs. Vehicle), and 3.3 in QVD-treated (p < 0.05 vs. Cisplatin). LDH release (%), a marker of cell membrane damage seen in necrosis was 7.1 in Vehicle-treated, 39.7 in Cisplatin-treated (p < 0.05 vs. Vehicle), and 13.5 in QVD-treated (p < 0.05 vs. Cisplatin). Caspase-1 activity and active caspase-1 protein (10 kDa) were significantly increased in Cisplatin-treated PTs. NLRP3 was strongly expressed in PTs, but there were no significant changes between groups. Pro-apoptotic BID (22 kDa) was unchanged between groups. IL-1α and IL-1β activity was increased in Cisplatin-treated PTs. QVD-OPH co-treatment decreased caspase-1, IL-1α, and IL-1β. In summary, caspase inhibition decreases caspase-1, IL-1α, and IL-1β but not NLRP3 or BID protein and protects against necrosis in cisplatin-treated freshly isolated PTs.