Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.

Enterotoxigenic Escherichia coli (ETEC) causes acute diarrhea and is transmitted through contaminated food and water; however, systematic procedures for its specific detection in foods have not been established. To establish an efficient detection method for ETEC in food, an interlaboratory study using ETEC O148 and O159 as representative serogroups was first conducted with 13 participating laboratories. A series of tests including enrichment, real-time PCR assays, plating on selective agars, and concentration by immunomagnetic separation followed by plating onto selective agar (IMS-plating methods) were employed. This study particularly focused on the detection efficiencies of real-time PCR assays for enterotoxin genes (sth, stp, and lt), IMS-plating methods, and direct plating onto sorbitol MacConkey agar and CHROMagar STEC medium, supplemented with tobramycin, which is a novel modification in the preparation of a selective agar. Cucumber and leek samples inoculated with ETEC O148 and O159, either at 4-7 CFU/25 g (low levels) or at 21-37 CFU/25 g (high levels) were used as samples with uninoculated samples used as controls. At high inoculation levels, the sensitivities of sth, stp, and lt detection, direct-plating, and IMS-plating methods in cucumber inoculated with O148 and in both foods inoculated with O159 were 100%. In leek inoculated with high levels of O148, the sensitivities of sth, stp, and lt detection, direct-plating, and the IMS-plating method were 76.9%, 64.1%, and 74.4%, respectively. At low inoculation levels, the sensitivities of sth, stp, and lt detection, direct plating, and IMS-plating method in cucumber inoculated with O148 and in both foods inoculated with O159 were in the range of 87.2-97.4%. In leek inoculated with low levels of O148, the sensitivities of sth, stp, and lt detection, direct plating, and the IMS-plating method were 59.0%, 33.3%, and 38.5%, respectively. Thus, ETEC in food contaminated with more than 21 CFU/25 g were detected at high rate (over 74%) using real-time PCR assays and IMS-plating onto selective agar. Therefore, screening sth, stp, and lt genes followed by isolation of STEC using the IMS-plating method may be an efficient method for ETEC detection.

Autonomous mechanisms of axon degeneration are frequently studied in vitro by mechanical axon injury of isolated sensory neurons. This has led to major advances in understanding the molecular pathways governing axon degeneration. However, this approach does not pay attention to potential glial mechanisms for the regulation of axon death. Here, I describe a straightforward protocol to seed purified rat Schwann cells on neuronal cultures in order to study the interaction between axons and these glia during axon degeneration.

BACKGROUND: Circulating tumor cells (CTC) play an important role in the screening and prognosis of lung cancer, but the low efficiency and specificity of CTC isolation obviously restrict its clinical application. The purpose of this study is to explore a new and efficient isolation method of CTC in patients with non-small cell lung cancer (NSCLC) in order to achieve the purpose of early diagnosis of NSCLC.
METHODS: Three kinds of immunolipid magnetic spheres of epidermal growth factor receptor (EGFR), vimentin and folic acid (FA) were prepared by thin film method. After characterization, the sorting scheme of cell line was explored, the optimal sorting scheme of NSCLC CTC was constructed, and its clinical application value was studied.
RESULTS: The average capture efficiency of EGFR, Vimentin and FA magnetic spheres used alone and in combination to lung cancer cell lines was 78%, 79%, 82% and 91%, respectively. In 60 patients with lung cancer, using 2 CTC per 7.5 mL blood as cutoff value, the positive rates of EGFR, Vimentin and FA magnets used alone and in combination were 65.0%, 33.3%, 93.3% and 100%, respectively. It was also found that the number of CTC detected by combined use of the three magnetic spheres was correlated with clinical stages (P<0.05).
CONCLUSIONS: The combination of three kinds of magnetic spheres can separate EGFR+, Vimentin+, FA+ expressed CTC, which is beneficial to the downstream analysis of CTC correlation. This study provides a new method to improve the efficiency of NSCLC CTC capture, and verifies that the captured CTC counting method can be used in the auxiliary diagnosis of lung cancer.
【中文题目：EGFR/Vimentin/Folic Acid磁球检测肺癌循环肿瘤细胞初探】 【中文摘要：背景与目的 循环肿瘤细胞（circulating tumor cell, CTC）在肺癌的筛查及预后方面发挥着重要的作用，但较低的CTC分离效率和特异性对其临床应用有着明显的制约，本研究旨在探讨非小细胞肺癌（non-small cell lung cancer, NSCLC）患者CTC的新型高效分离方法，以期达到对NSCLC的早期诊断的目的。方法 采用薄膜法制备表皮生长因子受体（epidermal growth factor receptor, EGFR）、波形蛋白（Vimentin）和叶酸（folic acid, FA）三种免疫脂质磁球，表征后通过细胞系进行分选方案的探索，构建对NSCLC CTC的最优分选方案，初步研究了其在临床上的应用价值。结果 EGFR、Vimentin和FA磁球磁球单独和联合使用对肺癌细胞株的平均捕获效率分别为78.0%、79.0%、82.0%和91.0%；在60例肺癌患者中，以每7.5 mL血液2个CTC为cutoff值，EGFR、Vimentin、FA磁球单独和联合使用阳性率分别为65.0%、33.3%、93.3%和100.0%，同时发现联合使用三种磁球检出的CTC数量与临床分期具有相关性（P<0.05）。结论 联合使用三种磁球可以分离EGFR+、Vimentin+和FA+表达且形态完整的CTC，有利于的CTC相关下游分析，本研究提供了一种提高NSCLC CTC捕获效率的新方法，且验证了捕获的CTC计数方法可用于肺癌的辅助诊断。】 【中文关键词：肺肿瘤；循环肿瘤细胞；EGFR；Vimentin；叶酸；免疫磁球】.

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.

Exosomes are cell-derived nanovesicles released into biological fluids, which are involved in cell-to-cell communication. The analysis of the content and the surface of the exosomes allow conclusions about the cells they are originating from and the underlying condition, pathology or disease. Therefore, the exosomes are currently considered good candidates as biomarkers to improve the current methods for clinical diagnosis, including cancer. However, due to their low concentration, conventional procedures for exosome detection including biosensing usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. In this paper, the immunomagnetic separation is presented as an alternative method for the specific isolation of exosomes in serum. To achieve that, a rational study of the surface proteins in exosomes, which can be recognized by magnetic particles, is presented. The characterization was performed in exosomes obtained from cell culture supernatants of MCF7, MDA-MB-231 and SKBR3 breast cancer cell lines, including TEM and nanoparticle tracking analysis (NTA). For the specific characterization by flow cytometry and confocal microscopy, different commercial antibodies against selected receptors were used, including the general tetraspanins CD9, CD63 and CD81, and cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). The effect of the serum matrix on the immunomagnetic separation was then carefully evaluated by spiking the exosomes in depleted human serum. Based on this study, the exosomes were preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further magnetic actuation on the surface of the electrode for the electrochemical readout. The performance of this approach is discussed and compared with classical characterization methods.

Diabetic nephropathy is associated with injury and loss of podocytes, specialized epithelial cells that are critical for glomerular filtration. This chapter describes a method of isolating and culturing podocyte cells from mouse adult kidneys. In this way, podocytes with genetic modifications can be obtained from transgenic animals and they can be used to study the effects of the diabetic environment in vitro.

Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.

The aim of this study was to evaluate the occurrence of Cryptosporidium oocysts in a drinking water treatment plant (DWTP) located in a rural area of northern Italy. Influent and effluent samples were collected at the DWTP over three years (2013-2016). In parallel, tap water samples from a public drinking fountain were collected as well. All samples were analyzed for the presence of Cryptosporidium spp. oocysts by a common method based on an immunomagnetic separation (IMS)/immunofluorescence assay (IFA), complemented by 4,6-diamidino-2-phenylindole (DAPI) staining. A reverse transcriptase-PCR (RT-PCR) protocol was added to evaluate oocyst viability. The results highlighted a high variability of oocyst concentrations across all samples (mean 4.3 ± 5.8/100 L) and a high variability in the percentage of DAPI-positive specimens (mean 48.2% ± 40.3%). Conversely, RT-PCR did not reveal the presence of viable C. parvum and C. hominis oocysts. A nested PCR targeting Cryptosporidium 18S ribosomal DNA, carried out in two water samples, confirmed the presence of a Cryptosporidium genotype associated with wild animals in the river and in tap water. The results obtained underline the vulnerability of the investigated surface water to Cryptosporidium spp. contamination. Although the recovered Cryptosporidium genotype is not a human pathogen, its presence demonstrates the existence of a potential pathogen Cryptosporidium spp. contamination risk. Moreover, these results underline the importance of also considering unconventional (not bacterial) biological contaminations (protozoa) in water resources in rural areas, including those of developed countries.

We assessed CTCs counts in NMCRC patients using four different techniques.
CTCs were detected in 63 NMCRC patients, 40 benign bowel diseases (BBD) and 40 normal controls (NC) using, flow-cytometry (FCM), CellSearch (CS), cytomorphology and quantitative real time (qPCR) for CK19, MUC1, CD44, CD133, ALDH1 expression. Results were correlated to progression free (PFS) and overall (OS).
Positive CTCs (≥4 cells /7.5 mL blood) were detected in 50.8% (32/63) NMCRC by FCM and 7.5% (3/40) BBD (p < .001). CTCs were detected in 34/63 (54%) NMCRC, 4/40 (10%) BBD (p < .001) by CS. CK19, MUC1, CD44, CD133 and ALDH1 were expressed in 35 (55.6%), 29 (46.0%), 28 (44.4%), 26 (41.3%) and 25 (41.3%) cases of NMCRC. In BBD 4/40 (10%) cases expressed CK19, MUC1 and CD44, while 2/40 (5%) expressed CD133. Cytomorphology showed the lowest sensitivity (47.6%) and specificity (90%) for CTCs detection. The combined use of FCM or CS with CTCs-mRNA markers improved the sensitivity and specificity to 68.3%, and 95.0%; respectively. Positive CTCs and mRNA markers expression were significantly associated with shorter 5-yr PFS and OS. In multivariate analysis, CTCs mRNA markers were independent prognostic factors for PFS and OS.
Enumeration of CTCs by FCM and RNA expression for specific colon cancer markers are comparable to CS regarding sensitivity and specificity. CTCs also represent novel therapeutic targets for NMCRC cases.