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To explore the methods of screening and biological characteristics of lung cancer stem cells. We selected the ABCG2 and ABCG2 cells from SPC-A-1/adriamycin(ADM)cell line induced by ADM and analyzed the tumorigenicity of ABCG2 and ABCG2 cells by flow cytometry and transplantation in nude mice. The average fluorescence intensity of SPC-A-1 cells was(1.001±0.014)×10 ,which was significantly lower than that of SPC-A-1/ADM cells [(10.257±0.023) ×10 ](=17.320,=0.001);the difference was also statistically significant between the ABCG2/BCRP-FITC treatment group and the SPC-A-1 control group(=5.269,=0.021) and the SPC-A-1 control group(=6.869, =0.012) and between the SPC-A-1/ADM cell control group and the SPC-A-1/ADM cell homotype control group(=8.112,=0.015).The positive rate of SPC-A-1/ADM cells treated with ABCG2/BCRP-FITC was 9.8%,39.84 times higher than that of SPC-A-1 cells;it showed significant difference between the ABCG2/BCRP-FITC group and the SPC-A-1/ADM group(=9.120,=0.005) and the SPC-A-1/ADM group(=8.257,= 0.006).The positive rate of group B cells was 684 times that of group A cells,and the difference was statistically significant(=11.235,=0.001),and the fluorescence intensity of group B cells was strong.The average tumorigenic volume of the mice inoculated with SPC-A-1 cells,group A cells,and group B cells was(6.96±1.82),(6.70±2.55),and(9.17±2.41) mm ,respectively.Among them,group B was the highest,but there was no significant difference among these three groups(=2.362,=0.086).The tumorigenic rate of group B cells was 75.00%,which was significantly higher than that of SPC-A-1 cells and group A cells(=19.780,=0.002). ABCG2 cells from human lung adenocarcinoma SPC-A-1/ADM cell line can be isolated by ABCG2 antibody combined with immunomagnetic beads sorting method,and the tumor formation rate in nude mice can be observed to explore the identification and biological characterization of lung cancer stem cells.
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OBJECTIVE To establish a primary culture of neurons under low density conditions by immunomagnetic beads (1MB) based cell separation technology combined with astrocyte conditioned medium (A-CM). METHODS The cerebral hemispheres of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension for primary culture. After 10 d of culture, the microglia and other irrelevant cells were removed by constant temperature oscillation, while the purity of astrocytes was identified by immunofluorescence staining with anti-glial fibrillary acidic protein (GFAP) antibody. A-CM was prepared using cell cultures that met purity requirements. The cerebral cortex and hippocampus of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension. High-purity neurons were obtained by immunomagnetic beads based cell separation technology, and A-CM was applied to primary culture of neurons under low density conditions. Morphological parameters of neurons during culture were observed, and neurons were identified by immunofluorescence staining with anti-160 ku neurofilament antibody. RESULTS The purity of astrocytes was over 95% by cellular immunofluorescence staining identification, which met the cell purity requirements of subsequent experiments. Morphological observation and cellular immunofluorescence staining of the mouse cortex and hippocampal neurons showed that the neurons grew well under low density culture conditions with high purity, and the morphological characteristics were distinct at different developmental stages. CONCLUSION The primary culture of the mouse cerebral cortex and hippocampal neurons under low density conditions is successfully established.
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Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.
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Humans , Male , Cell Separation , DNA , Immunomagnetic Separation , Lipocalins , Polymerase Chain Reaction , SpermatozoaABSTRACT
Objective:To study the signal enhancement of lung adenocarcinoma nude mice after injection of immunomagnetic bead solution (magnetic beads conjugated with monoclonal antibody NJ001) in micro-CT scan. Methods:The models of lung adenocarcino-ma nude mice were established by injecting SPC-A1-luc cells through the tail vein and were validated by bioluminescence imaging (BLI). The nude mice were divided into three groups: physiological saline group, bare magnetic bead group, and immunomagnetic bead group. Three groups of nude mice were injected with physiological saline, 750 nm bare magnetic bead solution, and immuno-magnetic bead solution via the tail vein every week, and micro-CT scan was taken before and 4 h after injection. Immunohistochemis-try (IHC) was used to detect the expression of antigen SP70 in tumor tissues. Results:The tumor was detected in the immunomagnetic bead group at the fourth week, whereas in the physiological saline and bare magnetic bead groups, the tumor was undetectable until the sixth week. The tumor intensities detected at the sixth week by micro-CT scan in the physiological saline, bare magnetic bead, and immunomagnetic bead groups were 59.05 ± 0.66, 60.69 ± 0.55, and 58.25 ± 0.32 before injection and 60.30 ± 1.83, 61.05 ± 0.68, and 67.41±3.82 after injection, respectively. Compared with the tumor intensities before injection, they significantly increased after injec-tion in the immunomagnetic bead group;the difference was statistically significant (P=0.0079). By contrast, no statistical significance was observed in the tumor intensities before and after injection in the physiological saline and bare magnetic bead groups (P=0.1867 and P=0.3839, respectively). Conclusion:The immunomagnetic beads had enhanced effect on micro-CT scan of lung adenocarcinoma nude mouse models.
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Objective To establish a near‐infrared fluorescent dye‐immunomagnetic beads coupling method for quantitative de‐tection of Mycobacterium tuberculosis early secretory antigenic target‐6(ESAT‐6) .Methods Near‐infrared fluorescent dye ,dylight 800 ,was used to mark ESTA‐6‐targeting monoclonal antibodies ,and the surface of nano‐magnetic beads were coated with ESAT‐6‐targeting polyclonal antibodies .Double antibody sandwich method was used for magnetic separation of conjugates and dissociants . Portable high‐sensitivity and low‐noise excitation fluorescence detector was used to detect the intensity of magnetic combination ,so as to test the ESAT‐6 content in test samples .Results The detecting linear range of this method was 2 .4-750 .0 ng/mL ,and the minimum detection limit was 0 .48 ng/mL .The recovery rate was 96% at a concentration of 10 ng/mL ,and at a concentration of 50 ng/mL the recovery rate was 95% .The between‐run coefficient of variation(CV)value was 5 .8% ,and the within‐run CV value was 4 .3% .The specificity and sensitivity of this method for detecting ESAT‐6 in clinical pleural effusion samples were 80% and 95% , respectively .Conclusion This method might have wide linear range ,high sensitivity and good stability in the detection of ESAT‐6 .
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Objective To establish a novel method by integrating immunomagnetic bead enrich-ment with immunochromatography for the detection of influenza A virus. Methods The immunomagnetic beads were prepared by using EDC/NHS method and then coupled with monoclonal antibodies against influ-enza A virus. A direct immunomagnetic beads-based immunochromatography for the detection of influenza A virus was developed by using double-antibody sandwich method and immunochromatography, which was fur-ther combined with immunomagnetic separation to establish the novel integrated method of immunomagnectic bead enrichment and immunochromatography. Clinical throat swab samples collected from patients with influ-enza A virus infection and healthy subjects were analyzed by the novel method and the results were compared with those by using the conventional colloidal gold immunochromatography to evaluate the specificity, sensi-tivity and positive coincidence rate of this established method. Results The direct immunomagnetic beads-based immunochromatography and the colloidal gold immunochromatography showed no significant differences in specificity and sensitivity and could be used to identify influenza A virus-positive samples with cycle threshold ( Ct) values less than or equal to 22 obtained by real-time PCR assay. The integrated method could identify positive samples with Ct values less than or equal to 28, indicating that the novel method was more sensitive. Conclusion The novel method by integrating immunomagnetic bead enrichment with immunochroma-tography was successfully established and suitable for the rapid and on-site detection of influenza A virus.
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Adipose-derived stem cells ( ASCs ) as potential seeded cells have been widely used in tissue engineering.Thus to obtain enough, high activity, high purity adipose-derived stem cells is the particular important premise of the application in tissue engineering.In this paper, the isolation and purification methods of ASCs were reviewed and the merit and demerit of different methods were compared in order to provide theoretical basis for safe and high-effective isolation and purification of ASCs.
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Objective To make a comparison for the neutrophils prepared either by induction of differentiation of myeloid leuke-mia cell line,or by separation and purification of peripheral blood cells,or by induction of myeloid differentiation of peripheral blood stem cells.Methods NB4 cells were induced differentiation by 1μmol/L all-trans retinoic acid (ATRA)to mature granulo-cytes;neutrophils were separated and purified from peripheral blood by lysis of red blood cells followed by negative selection using magnetic bead-labeled antibodies;hematopoietic stem cells were separated and purified from peripheral blood by Percoll gradient centrifugation followed by negative selection using magnetic bead-labeled antibodies,and were induced to myeloid differentiation by GM-CSF and G-CSF.Morphology and purity of neutrophils prepared by these three methods were studied by means of MGG stai-ning.CD18 protein expression and subcellular distribution were studied by means of immunofluorescence staining.Results Purity of neutrophil was above 40% by induction of differentiation of NB4 cells,and was about 90% if purified from peripheral blood,and was above 70% if induced by myeloid differentiation of peripheral blood stem cells.There was no obvious difference for CD18 ex-pression in neutrophils prepared by these three methods,and staining of CD18 had a dotted pattern distributed in these cells.Con-clusion Peripheral blood neutrophils prepared by lysis of red blood cells followed by negative selection using magnetic bead-labeled antibodies are with high purity and viability which is suitable for immediate test of neutrophils from fresh blood.Neutrophils pre-pared by myeloid differentiation of hematopoietic stem cell are with high viability and last for days,which can be used in long test for neutrphils.
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Objective To establish a high purity primary culture methods of human nasal epithelial cells (HNEC) in vitro and to provide a successful primary culture model for evaluation experiments of the nasal preparation .Methods Primary culture of human nasal epithelial cells were performed with enzymatic dissociation of isolated tissue and cultured in serum-free medium .HNEC were separated through magnetic field by immunomagnetic beads .We determined the purity of the separated cells by light microscopy and flow cytometry .The morphology of HNEC was observed with a scanning electron microscope .Results Under an inverted phase microscope ,the cells morphology was paving stone shaped .Under the scanning electron microscopy ,abundant microvilli and cilia differentiation were observed .Flow cytometry showed the epithelial cells accounted for 99% .Conclusion The highly purified HNEC can be directly isolated by the magnetic cell sorting system .The cell model can be used for the basic research of nasal cavity preparation .
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Objective To seek the specific receptors associated with hepatitis B virus (HBV) adhesion by separating the binding protein of the HBV preS1 region in HepG2 and performing the mass spectrometry .Methods The immunomagnetic bead method was adopted to separate HepG2 membrane protein combined with preS1 peptide fragment and the binding protein was separated by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ,then the destination strips was analyzed by LC-MS/MS mass spectrometry and retrieved by the database .Results 16 bands were separated from HepG2 membrane proteins combined with preS1 by SDS-PAGE ;14 kinds of proteins were identified from 6 bands with better repeatability separated from HepG 2 membrane proteins combined with preS1 .Conclusion Protein analyzed by the mass spectrometry is mainly related with the material transport , cellular signal transduction ,antigen presentation ,immune regulation and energy metabolism .
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ObjectiveTo explore the value of immuomagnetic beads(IMB) technique for detection of intraperitoneal free cancer cells from colorectal cancer.MethodsPeritoneal lavage fluid was obtained from 80 patients with colorectal cancer during laparotomy.Peritoneal lavage cytology (PLC) and IMB were used to detect free cancer cells in peritoneal lavage fluid.10 patients with hysteromyoma during laparotomy were enrolled into the control group.ResultsThe positive rate of PLC was 8.8％ (7/80),the positive rate of IMB was 28.8％ (23/80).The positive case after useing PLC detect,IMB detect also was positive.The detected samples of control group were negative by these two methods.IMB was superior to PLC ( x2 =10.503,P =0.001 ).ConclusionIMB was more sensitive and specific than PLC,which could provide a effective method for finding intraperitoneal free cancer cells.
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Background and purpose:Nowadays, the golden standard of diagnosis for malignant hydrothorax or ascites is exfoliocytology examination, but the missed diagnosis rate is too high. Other methods including immunologic test, telomerase activation test, conjugation of chromosome analysis with cytological examination test, and RT-PCR test. But none of them was widely used due to high cost or high false positive rate. Immunomagnetic beads (IMB) technique is a popular method all over the world in recent years. It was mainly used in isolating and purifying cells, but it was rarely used to detect cancer cells in patients with hydrothorax or ascites so far. Our aim was to fi nd an effi cient way to detect the cancer cells in patients with cancer related hydrothorax or ascites and to improve the corresponding diagnosis rate. Methods:In the experiments, both the traditional exfoliocytology examination method and IMB technique were used to detect the cancer cells in the hydrothorax or the ascites for comparison. Results:Using IMB technique and exfoliocytology method, the positive rates in 30 patients with cancers were 63.3% (19/30) and 23.3% (7/30), respectively, and the false positive rates in 30 patients without cancers were 3.3% (1/30) and 0.0% (0/30), respectively. It could be observed that the positive rate using IMB technique was much higher than that using exfoliocytology method (P0.05). Conclusions:The present study demonstrated that IMB technique is an accurate, sensitive, fast and economic method in detecting the cancer cells in patients with cancer related hydrothorax or ascites, especially for diagnosis and therapy in the early clinical stage. Due to the high effi ciency, IMB technique could be used after exfoliocytology examination to improve the diagnosis rate..
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Objective To isolate CESs by dynabeads coated with the specific antibody of CD146 from peripheral blood and its origin was identified.Methods One mL blood with or without admixed HUVECs was diluted(1∶2) in PBS-0.1% BSA.Anti-CD146-coated Dynabeads were added and incubated for 30mins at(4 ℃).Cells bound to anti-CD146-coupled beads were separated from blood in Dynal MPC and then washed and resuspended in(4 mL) buffer.After 4 additional washes with PBS-0.1% BSA using the magnet,the rosetted cells were flushed from the tube wall with (100 ?L) of PBS-BSA with acridine orange or Giemsa,and counted in a hemocytometer in a inverse phase contrast fluorescence microscope.Results The amount of CECs in healthy adult was 10.5(6～16.5)/mL(n=42).The recovery rate was 91%.Isolated CECs were confirmed by positive expression of vWF and CD31.Conclusion Isolation of CECs from blood by immunomagnetic beads coated with antibody of CD146 is characterized by its accuracy,time-saving,high recovery rate,less contaimination of blood and less damage to CECs.It can be used to quantify CECs and to analyze the functional performance of CECs from patients with vascular injury diseases.
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The objective of the experiment was to separate neural stem cells from fetal rat brain by using immunomagnetic beads, and to study their morphology and marker on cell membrane. Cell suspension of the fetal rat cerebral cortex was incubated with antibody against Nestin and the labeled cells were separated through magnetic field by immunomagnetic beads coated with the second antibody. We determined the purity of the separated cells and the cell activity by light microscopy and detected the Nestin expression ratio of the sorted cells by flow cytomety. The morphology of neural stem cells was observed with a scanning electron microscope. The results showed that the purity of the separated cells was 87 8%%+2 4%, the activity of the cells was 94 8%+2 3%, and the Nestin expression ratio of the sorted cells was 88 3%+1 04%. Binding of the sorted cells to beads was shown under scanning electron microscope.Our conclusions are the magnetic cell sorting system we used can effectively separate neural stem cells from the cerebral cortex cell suspension and maintain their activity. The sorted cells can be used in the subsequent experiments.
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Objective:To explore whether the Formula of Tonifying Kidney of Vital Essence can modulate secretion of IL-4 and IFN-? by the CD4+T cells from human decidua during first trimester,which will lead to further investigate the anti-abortional effect mechanisms of the Formula of Tonifying Kidney of Vital Essence.Methods:40 female adult SD rats were divided randomly into two groups:one group was gavaged with the Formula of Tonifying Kidney of Vital Essence;another was gavaged with normal saline,twice a day for 3 days.The two groups of rats were gavaged with the same volume of the decoction or saline.Thereafter the blood was collected from carotid artery.Human decidua tissues were collected by artificial abortion at 6-10 weeks of gestation.The CD4+ T cells were separated by MACS.CD4+ T cells were divided into four groups during cell culture.Group A was treated with 10% fetal calf serum,which was the control group.Group B was treated with 10% of the rat serum from the herbal medicine;Group C was treated with 20% of the rat serum from the herbal medicin.Group D was treated with the rat serum of the saline control.The supernatants were harvested at 24,48 and 72 h of the culture,respectively.The amounts of IL-4 and IFN-? in the culture supernatants were measured by ELISA.Results:In both group B and C of the culture the amount of IL-4 was evidently elevated when compared with the control,group A(P