We&#39;d like to thank the patients who volunteered their consent, time, and donation of blood samples. We also acknowledge Dr. Patrick Moriarty, Julie-Ann Dutton, and Mark McClellen at the Kansas University Medical Center for their collaboration and for implementing this protocol at a remote site. CY has received research support from NIH grant 1K08HL150271.

The study protocol was approved by the UCSD and KUMC Human Protections Program and conforms to the Declaration of Helsinki. All individuals provided informed consent for participation and blood collection.
1. Processing and cryopreservation of PBMCs
<ol>
	<li>One day prior to the blood collection, print out the checklist of materials and reagents listed in Table 2 and prepare and label accordingly. 
	NOTE: This protocol was designed for the collection o...

<ol>
	<li>Mathew, J., Sankar, P., Varacallo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physiology, Blood Plasma. , (2018).</li><li>Van Oost, B., Van Hien-Hagg, I. H., Timmermans, A. P., Sixma, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+thrombin+on+the+density+distribution+of+blood+platelets:+Detection+of+activated+platelets+in+the+circulation.">The effect of thrombin on the density distribution of blood platelets: Detection of activated platelets in the circulation.</a> Blood. 62 (2), 433-438 (1983).</li><li>Hassani, M., 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=On+the+origin+of+low-density+neutrophils.">On the origin of low-density neutrophils.</a> Journal of Leukocyte Biology. 107 (5), 809-818 (2020).</li><li>Norouzi, N., Bhakta, H. C., Grover, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+cells+by+their+density.">Sorting cells by their density.</a> PLOS ONE. 12 (7), e0180520 (2017).</li><li>Manzone, T. A., Dam, H. Q., Soltis, D., Sagar, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+volume+analysis:+A+new+technique+and+new+clinical+interest+reinvigorate+a+classic+study.">Blood volume analysis: A new technique and new clinical interest reinvigorate a classic study.</a> JNMT. 35 (2), 55-63 (2007).</li><li>Kleiveland, C. 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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. -. 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This protocol for PBMC collection and cryopreservation has been successfully implemented by individuals with and without prior research laboratory training. In our application, FACS and RNA-sequencing of highly viable monocytes purified from stored PBMCs resulted in high-quality sequences.
A major strength of this protocol is its accessibility. The technique presented in the protocol utilizes tubes pre-packed with a solid-density gradient medium. As a result, whole blood can be collected direc...

This protocol demonstrates an accessible method and workflow for the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood. This protocol is especially targeted towards novice research technicians, students, and clinical lab staff with the objective of facilitating the collection and cryopreservation of PBMCs without assuming prior training in laboratory techniques.
This protocol utilizes centrifugation to separate the components of whole blood by density. Whole blood consists of four main components listed in order of decreasing density: red blood cells/erythrocytes (~45% of volume), white blood cells (&#60;1% of volume), platelets (&#60;1% of volume), and plasma (~55% of volume)1,2,3,4,5,6. White blood cells can be subdivided into two categories based on their nuclei characteristics: round or multi-nucleated6. PBMCs are defined as white blood cells with round nuclei and consist of the following cell types: lymphocytes (T cells, B cells, NK cells), dendritic cells, and monocytes6. Multi-nucleated white blood cells include granulocytes, which consist of the following cell types: neutrophils, basophils, and eosinophils6. Multi-nucleated white blood cells are denser than PBMCs6. The densities of each component of whole blood are detailed in Table 1.
In this protocol, whole blood is collected in density gradient centrifugation tubes. These tubes contain a pre-packed density gradient medium that has a density of 1.077 g/mL. Following centrifugation, denser cells, including multi-nucleated white blood cells and erythrocytes, are separated from PBMCs and platelets by the density gradient medium (Figure 1A)6,7. The PBMC and platelet fraction is then collected, washed, and centrifuged to remove platelets. The resulting purified PBMCs are collected and stored at -80 &#176;C or in liquid nitrogen. Cryopreserved PBMCs may be viably thawed and directly used in downstream analyses or additionally processed to isolate specific component cell types.
This protocol has been optimized for high-quality RNA sequencing from highly viable PBMCs. In this article, PBMCs were isolated and cryopreserved from patients in an outpatient clinic. Subsequently, monocytes were isolated from PBMCs by FACS and analyzed via RNA-sequencing. However, the protocol can be widely adapted to other experimental needs such as cell culture, gene editing, ex-vivo functional studies, single-cell analyses, phenotyping by flow cytometry or cytometry by time of flight, isolation of DNA/RNA or proteins, slides for immunohistochemistry, amongst others8,9,10,11,12,13,14,15,16,17,18.
In addition to PBMC collection from density gradient centrifugation tubes, this protocol reviews how to collect plasma and buffy coat via centrifugation using an EDTA tube. After centrifugation, whole blood is separated into plasma, erythrocytes, and a thin interface layer termed buffy coat containing leukocytes (Figure 1B)6. The buffy coat is commonly used for DNA extraction and subsequent genomic analyses19,20. The plasma layer contains the cell-free components of whole blood and can be used for biomarker assays21,22.

<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

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.

Following PBMC collection and cryopreservation, viability of thawed PBMCs, monocytes, and lymphocytes from 56 unique samples was assessed by flow cytometry using reagents listed in Table 4 following manufacturers&#39; instructions (Figure 2A-F). A mean &#177; SD viability of PBMCs, monocytes, and lymphocytes of 94 &#177; 4.0%, 98 &#177; 1.1%, and 93 &#177; 5.6%, respectively, was achieved (Figure 2G). Viability measurement by Tr...

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

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

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

In this protocol, we aim to provide an easy-to-follow, step-by-step tutorial on how to collect and store high viability peripheral blood and mononuclear cells, or PBMCs, from whole blood. PBMCs include lymphocytes, dendritic cells, and monocytes. Bulk PBMCs can be processed to isolate individual cell types and are commonly used in a wide variety of experiments and assays, including RNA sequencing and flow cytometry.In addition to PBMCs, we also demonstrate how to collect EDTA plasma and whole buffy coat. Prior to beginning this protocol, please prepare the materials and reagents listed in Table 2. Do note that this protocol is designed for the collection of PBMCs from six density gradient centrifugation tubes, each of which yields approximately 12 million cells.Scale accordingly. Using standard phlebotomy technique, collect whole blood in six density gradient centrifugation tubes and one EDTA tube until filled. Centrifuge all tubes at 1800g for 20 minutes at room temperature.If needed, collected tubes may be stored up to four hours and then processed simultaneously. Refer to supplemental video 1 for a demonstration of how to operate a centrifuge. After centrifugation of the tube containing the density gradient medium, plasma and PBMCs will be above the density gradient medium.Erythrocytes and granulocytes will settle to the bottom. Refer to Figure 1A for a visualization. After centrifugation, carefully open the density gradient centrifugation tube.Use a transfer pipette to remove and discard the translucent yellow layer containing plasma. Do not disturb the hazy layer of cells directly above the density gradient medium. Err on the side of leaving excess plasma rather than discarding PBMCs.Repeat for all tubes. With a new transfer pipette, pipette the remaining volume containing cells and residual plasma in each tube up and down gently several times above the density gradient medium. After resuspension, pipette the resuspended contents of the tube into a labeled 50 mL conical tube.Repeat for all tubes. Fill the 50 mL tube up to the 50 mL line with solution 1 by pouring. Centrifuge the 50 mL tube at 300g for 10 minutes at room temperature.The plasma and buffy collection protocol detailed in Section 2 may be completed during this time. Following centrifugation, the pellet contains PBMCs and the supernatant contains platelets and residual plasma. Discard as much supernatant as possible.Tap as needed to remove residual drops. Pour a 15 mL tube of solution 2 into the 50 mL conical tube containing the PBMC pellet. Gently invert the tube to resuspend the pellet.Aspirate 15 mL of solution 3 using a transfer pipette. Add dropwise to the 50 mL tube. Solution 3 contains DMSO, which is toxic to cells at room temperature.Do not add solution 3 until ready to be immediately processed. Invert the tube gently three times to mix. Fill each pre-labeled and pre-chilled cryo vial with 1 mL of PBMCs using a multi-dispense pipette.Refer to supplemental video 2 for a demonstration of how to operate a multi-dispense pipette. Place the cryo vials inside a pre-chilled freezing container. Then, move the container to a negative 80 degree Celsius freezer.Keep the cryo vials inside the freezing container for a minimum of 12 hours. Then, transfer to a labeled storage box at negative 80 degrees Celsius. Alternatively, transfer the frozen cryo vials to liquid nitrogen for long-term storage.After centrifugation, the top layer consists of plasma, the bottom contains erythrocytes, and the interface will be the buffy coats. See Figure 1B for a visualization. Using a new transfer pipette, draw up as much plasma as possible without disturbing the buffy coat layer.Then, dispense 0.5 mL aliquots into pre-labeled cryo vials. Aspirate the buffy coat layer and transfer it to the appropriately labeled cryo vial. Note that some plasma and erythrocytes may contaminate the buffy coat collection.After collection, store the cryo vials in a negative 80 degree Celsius freezer. Alternatively, store in liquid nitrogen for long-term storage. Prior to starting the PBMC thawing protocol, please prepare the materials and reagents listed in Table 3.This protocol is designed for the thawing of one 1.5 mL tube containing approximately 2 million cells. Scale accordingly. Do note that the absolute number of cells may differ significantly between different patients and different treatment conditions.Transfer frozen PBMCs from storage using dry ice and thaw in a 37-degree Celsius incubator for about 4 minutes. If desired, an aliquot can be taken for cell counting and viability measurements as detailed in section 4 of the written protocol. After thawing, add 1 mL of solution 4 to each cryo vial.Then, pour the contents into a pre-labeled 50 mL tube containing 10 mL of solution 4 for every PBMC tube thawed. Tap to remove residual drops. Place the cell strainer on the empty 50 mL tube and then pour the cell suspension through the cell strainer.If needed, lightly tap to break the surface tension. Centrifuge each tube containing the strained cells at 400g for 7 minutes at room temperature. After centrifugation, pour out the supernatant containing DMSO.Tap as needed to remove residual drops. Resuspend the cell pellet in the desired medium appropriate for downstream experimental needs, such as cell culture media or flow cytometry antibody cocktails. Proceed with your experiment.Following PBMC collection and cryopreservation, viability of thawed PBMCs, monocytes, and lymphocytes from 56 unique samples was assessed by flow cytometry using reagents listed in Table 4 following manufacturer's instructions shown in Figures 2A through F.A mean and standard deviation viability of PBMCs, monocytes, and lymphocytes of 94 plus or minus 4%98 plus or minus 1.1%and 93 plus or minus 5.6%respectively, was achieved, shown in Figure 2G. Viability measurements by trypan blue exclusion yielded a mean viability of 88 plus or minus 7.5%and a cell count of 2.3 times 10 to the 6 plus or minus 1.9 times 10 to the 6. Flow cytometry analysis also demonstrated that live monocytes comprised 17 plus or minus 5.9%and lymphocytes made up of 53 plus or minus 13%of total PBMCs.Subsequently, monocytes were sorted from thawed PBMCs from 59 unique samples by flow cytometry and submitted for library preparation and RNA sequencing as described elsewhere. A mean and standard deviation total sequence count of 18 plus or minus 16.3 million was achieved with a 93 plus or minus 6.3%read alignment and 49.6 plus or minus 1.4 GC content as shown in Figures 3A and B.A mean and standard deviation uniquely mapped reads percentage of 88 plus or minus 3.6%was found with a subset of 56 unique samples. These parameters demonstrate that highly viable PBMCs suitable for downstream applications including high-quality RNA sequencing can be obtained by operators with any level of laboratory training using this protocol.When performing this protocol, it is critical to work quickly to avoid cell death, especially after addition of solution 3, which is toxic to cells at room temperature. In addition, the cells must also be handled gently to minimize damage. This protocol provides an efficient, easy-to-follow method for the isolation of PBMCs, EDTA plasma, and buffy coat.These immune cells can be used for a broad range of experiments and assays focused on the role of the immune system in different biological contexts.

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

Research

JoVE Journal

Medicine

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.