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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The authors have no relevant financial or non-financial interests to disclose.

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

1.4K visualizações.  University of California San Diego. Neste protocolo, pretendemos fornecer um tutorial passo a passo fácil de seguir sobre como coletar e armazenar células mononucleares e de sangue periférico de alta viabilidade, ou PBMCs, de sangue total. As PBMCs incluem linfócitos, células dendríticas e monócitos. Os PBMCs em massa podem ser processados para isolar tipos de células individuais e são comumente usados em uma ampla variedade de experimentos e ensaios, incluindo sequenciamento de RNA e ...