Name: isolation and cryopreservation of highly viable human peripheral blood mononuclear cells from whole blood: a guide for beginners
Uploaded: 2024-10-25T13:30:00
Description: Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This ...

<ol>
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D., Zola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+whole+mononuclear+cells+from+peripheral+blood+and+cord+blood.">Isolation of whole mononuclear cells from peripheral blood and cord blood.</a> Curr Protoc Immunol. 85 (1), 7.1.1-7.1.8 (2009).</li><li>Faass, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+heptose+metabolites+and+the+cag+pathogenicity+island+to+the+activation+of+monocytes/macrophages+by+Helicobacter+Pylori.">Contribution of heptose metabolites and the cag pathogenicity island to the activation of monocytes/macrophages by Helicobacter Pylori.</a> Front Immunol. 12, e632154 (2021).</li><li>Preobrazhensky, S. N., Bahler, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomagnetic+bead+separation+of+mononuclear+cells+from+contaminating+granulocytes+in+cryopreserved+blood+samples.">Immunomagnetic bead separation of mononuclear cells from contaminating granulocytes in cryopreserved blood samples.</a> Cryobiology. 59 (3), 366-368 (2009).</li><li>Schenz, J., Obermaier, M., Uhle, S., Weigand, M. A., Uhle, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+granulocyte+contamination+from+peripheral+blood+mononuclear+cells+of+patients+with+sepsis+and+how+to+remove+it+-+A+technical+report.">Low-density granulocyte contamination from peripheral blood mononuclear cells of patients with sepsis and how to remove it - A technical report.</a> Front Immunol. 12, e684119 (2021).</li><li>Tessema, B., Riemer, J., Sack, U., K&#246;nig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+stress+assay+in+peripheral+blood+mononuclear+cells:+factors+influencing+its+results.">Cellular stress assay in peripheral blood mononuclear cells: factors influencing its results.</a> Int J Mol Sci. 23 (21), 13118 (2022).</li><li>Chen, X., Thibeault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2013+35th+Annual+International+Conference+of+the+IEEE+Engineering+in+Medicine+and+Biology+Society+(EMBC).">2013 35th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC).</a> IEEE. , 6228-6231 (2013).</li><li>Chen, X., Thibeault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=abc.">abc.</a> , 6228-6231 (2013).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+storage+temperature+on+PBMC+gene+expression.">The effects of storage temperature on PBMC gene expression.</a> BMC Immunol. 17, 6 (2016).</li><li>Ticha, O., Moos, L., Bekeredjian-Ding, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+long-term+cryopreservation+of+PBMC+on+recovery+of+B+cell+subpopulations.">Effects of long-term cryopreservation of PBMC on recovery of B cell subpopulations.</a> J Immunol Methods. 495, e113081 (2021).</li><li>Chao, S. -. H., Price, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavopiridol+inactivates+P-TEFb+and+blocks+most+RNA+polymerase+II+transcription+in+vivo.">Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo.</a> J of Biol Chem. 276 (34), 31793-31799 (2001).</li><li>Banfi, G., Salvagno, G. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 &#181;L Tips</td>
			<td>Gilson</td>
			<td>F174501</td>
			<td>Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Biopioneeer</td>
			<td>CNT-15</td>
			<td>To hold Solutions 2/3 
			2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>2 mL Cryovials</td>
			<td>Globe Scientific</td>
			<td>3012</td>
			<td>Approximately 40 cryovials needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>20 &#181;L Tips</td>
			<td>Gilson</td>
			<td>F174201</td>
			<td>For use in cell counting; volume needed is dependent on method of cell counting used. 1 tip is needed per 1 mL of unpooled frozen PBMCs to be defrosted</td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>CEM Corporation</td>
			<td>50-187-7683</td>
			<td>2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection 
			3 needed per 1 mL of unpooled frozen PBMCs to be defrosted</td>
		</tr>
		<tr>
			<td>CD14 Antibody</td>
			<td>Biolegend</td>
			<td>325621</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16 Antibody</td>
			<td>Biolegend</td>
			<td>360723</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19 Antibody</td>
			<td>Biolegend</td>
			<td>363007</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 Antibody</td>
			<td>Biolegend</td>
			<td>300405</td>
			<td></td>
		</tr>
		<tr>
			<td>CD56 Antibody</td>
			<td>Biolegend</td>
			<td>318303</td>
			<td></td>
		</tr>
		<tr>
			<td>CD66b Antibody</td>
			<td>Biolegend</td>
			<td>305103</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Biopioneeer</td>
			<td>DGN258367</td>
			<td>1 needed per 1 mL of unpooled frozen PBMCs to be defrosted</td>
		</tr>
		<tr>
			<td>CPT Mononuclear Cell Preparation Tube</td>
			<td>BD Biosciences</td>
			<td>362753</td>
			<td>6 tubes needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>Cryo Freezer Box</td>
			<td>Southern Labware</td>
			<td>SB2CC-81</td>
			<td>Holds 81 tubes 
			1 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>Cryotube Rack</td>
			<td>Fisherbrand</td>
			<td>05-669-45</td>
			<td>Holds up to 50 cryovials</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td>Approximately 2.5 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>DNase/RNase-Free Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td>Approximately 2 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection 
			Approximately 9 mL needed per 1 mL of frozen PBMCs to be defrosted</td>
		</tr>
		<tr>
			<td>EDTA Tubes</td>
			<td>BD Biosciences</td>
			<td>366643</td>
			<td>1 tube needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>Flavopiridol</td>
			<td>Sigma-Aldrich</td>
			<td>F3055-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-DR Antibody</td>
			<td>Biolegend</td>
			<td>307609</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>GeminiBio</td>
			<td>800-120</td>
			<td>Approximately 25 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>Human Trustain FcX</td>
			<td>Biolegend</td>
			<td>422301</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>W292912-1KG-K</td>
			<td>For use in Mr. Frosty</td>
		</tr>
		<tr>
			<td>Label Printer</td>
			<td>Phomemo</td>
			<td>M110-WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Live-Dead Stain</td>
			<td>Biolegend</td>
			<td>423105</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td>Holds up to 18 tubes. 
			2 Mr. Frostys needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>Multidispense Pipette</td>
			<td>Brandtech</td>
			<td>705110</td>
			<td></td>
		</tr>
		<tr>
			<td>Multidispense Pipette Tips</td>
			<td>Brandtech</td>
			<td>705744</td>
			<td>Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>P1000 Pipette</td>
			<td>Gilson</td>
			<td>F144059M</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Pipette</td>
			<td>Gilson</td>
			<td>F144056M</td>
			<td>For use in cell counting; volume needed is dependent on method of cell counting used.</td>
		</tr>
		<tr>
			<td>Printer Labels</td>
			<td>Phomemo</td>
			<td>PM-M110-3020</td>
			<td>Approximately 45 labels needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection 
			Approximately 5 labels needed per 1 mL of unpooled frozen PBMCs to be defrosted</td>
		</tr>
		<tr>
			<td>RNase-Free EDTA (0.5 M)</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td>Approximately 40 &#181;L needed per 1 mL of frozen PBMCs to be defrosted</td>
		</tr>
		<tr>
			<td>RNase-Free PBS (10X)</td>
			<td>Invitrogen</td>
			<td>AM9625</td>
			<td>Approximately 1 mL needed per 1 mL of frozen PBMCs to be defrosted</td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td>N2111</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td>Approximately 80 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>Transfer Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-711-7M</td>
			<td>Approximately 5 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection</td>
		</tr>
		<tr>
			<td>Tube Holder</td>
			<td>Endicott-Seymour</td>
			<td>14-781-15</td>
			<td>Holds up to 80 CPT/EDTA Tubes</td>
		</tr>
	</tbody>
</table>

isolation and cryopreservation of highly viable human peripheral blood mononuclear cells from whole blood: a guide for beginners

Tam Kandan Yüksek Derecede Canlı İnsan Periferik Kan Mononükleer Hücrelerinin İzolasyonu ve Dondurularak Saklanması: Yeni Başlayanlar İçin Bir Kılavuz

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This ...

isolation-cryopreservation-highly-viable-human-peripheral-blood

Research

JoVE Journal

Medicine

Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Tüm Kan enoksaparin Antikoagülan Etkinliğinin Rapid Point-of-Care Assay

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Tıp

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

İnsan Tüm Kan gerçek zamanlı sitotoksisite deneyleri

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Topluluk tabanlı Ayar Elde Edilen Örneklerin hızlı Fraksiyonlama ve Tam Kan Bileşenlerinin İzolasyonu

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

II-45 singenik Rat Mezotelyoma Modelinde Ortotopik Yerleştirilmesi ve Periferik Bağışıklık Hücre İzleme

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Trombosit transfüzyonları Microparticles için yöntem eleme rutin

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Farklı Homojenizasyon Yöntemleri ile Fare Beyin/Omurilikten Alınan Çoklu Hücre Tiplerinin Eşzamanlı Akış Sitometrik Karakterizasyonu

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1&#8211;blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1&#8211;blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1&#8211;blocking antibodies in cancer patients.

We developed a simple flow cytometry assay for evaluating the binding of PD-1&#8211;blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

Periferik Kan Damlasından Elde Edilen T Hücrelerine Bağlı Pd-1-Blokaj Antikorlarının İzlenmesi

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.