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 of 6 density gradient centrifugation tubes. Each tube yields roughly 12 million PBMCs, of which approximately 50% and 20% will be live lymphocytes and monocytes respectively. Scale accordingly for experimental needs. Note that the absolute number of cells may differ significantly between patients or treatment conditions.</li>
	<li>Using the standard phlebotomy technique, collect whole blood in 6 density gradient centrifugation tubes and 1 EDTA tube until filled, approximately 6 mL of volume. 
	NOTE: If samples from multiple patients or treatment conditions are required, the collected tubes may be stored up to 4 h and then processed simultaneously.</li>
	<li>After collection, centrifuge all tubes at 1,800 x g for 20 min at room temperature. 
	NOTE: Refer to Supplemental Video 1 for a demonstration of how to operate a centrifuge.</li>
	<li>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 caution, leaving excess plasma rather than discarding PBMCs. Repeat for all tubes. 
	NOTE: 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.</li>
	<li>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.</li>
	<li>After resuspension, pipette the resuspended contents of the tube into a labeled 50 mL conical tube. Repeat for all tubes.</li>
	<li>Pour Solution 1 (RPMI Medium) into the 50 mL tube until it reaches the 50 mL line.</li>
	<li>Repeat steps 1.4 to 1.7&#160;for each patient or treatment condition as needed. 
	NOTE: Ensure that different samples are not mixed and use a new transfer pipette to avoid contamination.</li>
	<li>Centrifuge the 50 mL tube at 300 x g for 10 min at room temperature. 
	NOTE: The plasma and buffy collection protocol detailed in Section 2 may be completed during this time.</li>
	<li>Discard as much supernatant as possible. Tap as needed to remove residual drops. 
	NOTE: Following centrifugation, the pellet contains PBMCs, and the supernatant contains platelets and residual plasma.</li>
	<li>Pour a 15 mL tube of Solution 2 (RPMI Medium + 12.5% Human Serum Albumin + 1 &#181;M Flavopiridol) into the 50 mL conical tube containing the PBMC pellet. Gently invert the tube to resuspend the cell pellet.</li>
	<li>Aspirate 15 mL of Solution 3 (RPMI Medium + 11.25% Human Serum Albumin + 1 &#181;M Flavopiridol + 10% DMSO) using a transfer pipette. Add dropwise to the 50 mL tube. 
	NOTE: Solution 3 contains DMSO, which is toxic to cells at room temperature. Do not add Solution 3 until it is ready to be immediately processed.</li>
	<li>Invert the tube gently 3 times to mix.</li>
	<li>Fill each pre-labeled and pre-chilled cryovial with 1 mL of PBMCs using a multi-dispense pipette. 
	NOTE: Refer to Supplemental Video 2 for a demonstration of how to operate a multi-dispense pipette.</li>
	<li>Place the cryovials inside a pre-chilled freezing container. Then, move the container to a -80 &#176;C freezer.</li>
	<li>Repeat steps 1.9 to 1.13 for each patient or treatment condition as needed.</li>
	<li>Keep the cryovials inside the freezing container for a minimum of 12 h, then transfer them to a labeled storage box at -80 &#176;C. 
	NOTE: Alternatively, transfer the frozen cryovials to liquid nitrogen (&#60;-135 &#176;C) for long-term storage.</li>
</ol>
2. Processing of plasma and buffy coat from EDTA tubes
<ol>
	<li>Centrifuge at 1,800 x g for 10 min at room temperature.&#160; 
	NOTE: After centrifugation, the top layer consists of plasma, the bottom contains erythrocytes, and the interface will be the buffy coat. See Figure 1B for a visualization.</li>
	<li>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 prelabeled cryovials. Aspirate the buffy coat layer and transfer it to the appropriately labeled cryovial. 
	NOTE: Some plasma and erythrocytes may contaminate the buffy coat collection.</li>
	<li>Repeat step&#160;2.2 for each patient or treatment condition as needed.</li>
	<li>After collection, store the cryovials in a -80 &#176;C freezer. 
	NOTE: Alternatively, store in liquid nitrogen (&#60;-135 &#176;C) for long-term storage.</li>
</ol>
3. Thawing PBMCs
<ol>
	<li>One day before thawing, print out a checklist of materials and reagents listed in Table 3 and prepare and label accordingly. 
	NOTE: This protocol was designed for the thawing of 1 1.5 mL tube containing 1 mL of PBMCs. Each tube yields approximately 2 million PBMCs, of which approximately 50% and 20% will be live lymphocytes and monocytes, respectively. Scale accordingly for experimental needs. Note that the absolute number of cells may differ significantly between patients or treatment conditions.</li>
	<li>Transfer frozen PBMCs from storage using dry ice and thaw in a 37 &#176;C incubator for 4 min, or just prior to the last ice crystal thawing. 
	NOTE: If desired, an aliquot can be taken for cell counting and viability measurements, as detailed in Section 4.</li>
	<li>After thawing, add 1 mL of Solution 4 (PBS + 2 mM EDTA) to each cryovial. 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.</li>
	<li>Place the cell strainer on the empty 50 mL tube and then pour the cell suspension through the cell strainer. 
	NOTE: If needed, lightly tap to break the surface tension.</li>
	<li>Centrifuge each tube containing the strained cells at 400 x g for 7 min at room temperature.</li>
	<li>After centrifugation, pour out the supernatant containing DMSO. Tap as needed to remove residual drops.</li>
	<li>Resuspend the cell pellet in the desired medium appropriate for downstream experimental needs (e.g., cell culture media, flow cytometry antibody cocktail, etc.).</li>
</ol>
4. Cell counting and viability
<ol>
	<li>Immediately after thawing frozen PBMCs in step 3.2, pipette 20 &#181;L of cells into a 1.5 mL tube. Add an equal volume of 0.4% Trypan Blue solution and pipette up and down gently 3-4 times to mix.</li>
	<li>Using an automated cell counter, follow the manufacturer&#39;s protocol to count cells and assess viability. Ensure that the dilution factor of 2 is accounted for. 
	NOTE: As described elsewhere, a hemocytometer may be used as an alternative method for assessing cell count and viability23.</li>
</ol>

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The impact of food bioactives on health: In vitro and ex vivo models. , 161-167 (2015).</li><li>Rosado, 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=Advances+in+biomarker+detection:+Alternative+approaches+for+blood-based+biomarker+detection.">Advances in biomarker detection: Alternative approaches for blood-based biomarker detection.</a> Adv Clin Chem. 92, 141-199 (2019).</li><li>Riedhammer, C., Halbritter, D., Weissert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Multiple sclerosis: Methods and Protocols. 1304, 53-61 (2016).</li><li>Panda, S. K., Ravindran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Culture+of+Human+PBMCs.">In vitro Culture of Human PBMCs.</a> Bio Protoc. 3 (3), e322 (2013).</li><li>He, D., 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=Whole+blood+vs+PBMC:+Compartmental+differences+in+gene+expression+profiling+exemplified+in+asthma.">Whole blood vs PBMC: Compartmental differences in gene expression profiling exemplified in asthma.</a> Allergy Asthma Clinic Immol. 15, 67 (2019).</li><li>Pel&#225;k, O., 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=Lymphocyte+enrichment+using+CD81-targeted+immunoaffinity+matrix.">Lymphocyte enrichment using CD81-targeted immunoaffinity matrix.</a> Cytometry Part A. 91 (1), 62-72 (2017).</li><li>Higdon, L. E., 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=Impact+on+in-depth+immunophenotyping+of+delay+to+peripheral+blood+processing.">Impact on in-depth immunophenotyping of delay to peripheral blood processing.</a> Clin Exp Immunol. 217 (2), 119-132 (2024).</li><li>Liu, Z., 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=Photoactivatable+aptamer-CRISPR+nanodevice+enables+precise+profiling+of+interferon-gamma+release+in+humanized+mice.">Photoactivatable aptamer-CRISPR nanodevice enables precise profiling of interferon-gamma release in humanized mice.</a> ACS Nano. 18 (4), 3826-3838 (2024).</li><li>Ren, Z., Li, C., Wang, J., Sui, J., Ma, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptome+revealed+dysregulated+RNA-binding+protein+expression+patterns+and+functions+in+human+ankylosing+spondylitis.">Single-cell transcriptome revealed dysregulated RNA-binding protein expression patterns and functions in human ankylosing spondylitis.</a> Front Med. 11, 1369341 (2024).</li><li>Honardoost, M. A., 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=Systematic+immune+cell+dysregulation+and+molecular+subtypes+revealed+by+single-cell-RNA-seq+of+subjects+with+type+1+diabetes.">Systematic immune cell dysregulation and molecular subtypes revealed by single-cell-RNA-seq of subjects with type 1 diabetes.</a> Genome Med. 16, 45 (2024).</li><li>Amini, A., Esmaeili, F., Golpich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+role+of+lncRNAs+in+amelioration+of+Parkinson's+disease+symptoms+by+transplantation+of+dopaminergic+cells.">Possible role of lncRNAs in amelioration of Parkinson's disease symptoms by transplantation of dopaminergic cells.</a> NPJ Parkinsons Dis. 10, 56 (2024).</li><li>Luo, Y., 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=Sars-Cov-2+spike+induces+intestinal+barrier+dysfunction+through+the+interaction+between+CEACAM5+and+Galectin-9.">Sars-Cov-2 spike induces intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9.</a> Front Immunol. 15, e1303356 (2024).</li><li>Zhang, X., Wu, G., Ma, X., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+alterations+and+PI3k-PKB+pathway+suppression+in+patients+with+allergic+rhinitis+undergoing+sublingual+immunotherapy.">Immune cell alterations and PI3k-PKB pathway suppression in patients with allergic rhinitis undergoing sublingual immunotherapy.</a> Adv Ther. 41 (2), 777-791 (2024).</li><li>Rucci, C., 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=Exploring+mitochondrial+DNA+copy+number+in+circulating+cell-free+DNA+and+extracellular+vesicles+across+cardiovascular+health+status:+A+prospective+case-control+pilot+study.">Exploring mitochondrial DNA copy number in circulating cell-free DNA and extracellular vesicles across cardiovascular health status: A prospective case-control pilot study.</a> FASEB J. 38 (10), e23672 (2024).</li><li>Ghamrawi, R., 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=Buffy+coat+DNA+Methylation+Profile+is+Representative+of+Methylation+Patterns+in+White+Blood+Cell+Types+in+Normal+Pregnancy.">Buffy coat DNA Methylation Profile is Representative of Methylation Patterns in White Blood Cell Types in Normal Pregnancy.</a> Front Bioeng Biotechnol. 9, e782843 (2022).</li><li>Johansson, C., 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=Plasma+biomarker+profiles+in+autosomal+dominant+Alzheimer's+disease.">Plasma biomarker profiles in autosomal dominant Alzheimer's disease.</a> Brain. 146 (3), 1132-1140 (2023).</li><li>Bonaterra-Pastra, A., 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=Comparison+of+plasma+lipoprotein+composition+and+function+in+cerebral+amyloid+angiopathy+and+Alzheimer's+disease.">Comparison of plasma lipoprotein composition and function in cerebral amyloid angiopathy and Alzheimer's disease.</a> Biomedicines. 9 (1), 72 (2021).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cell+number+by+hemocytometry+counting.">Estimation of cell number by hemocytometry counting.</a> Cold Spring Harb Protoc. (11), prot097980 (2019).</li><li>Cobo, I., 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=Monosodium+urate+crystals+regulate+a+unique+JNK-dependent+macrophage+metabolic+and+inflammatory+response.">Monosodium urate crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response.</a> Cell Rep. 38 (10), e110489 (2022).</li><li>Corkum, C. P., 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=Immune+cell+subsets+and+their+gene+expression+profiles+from+human+PBMC+isolated+by+Vacutainer+Cell+Preparation+Tube+(CPT&#8482;)+and+standard+density+gradient.">Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT&#8482;) and standard density gradient.</a> BMC Immunology. 16, 48 (2015).</li><li>Golke, T., 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=Delays+during+PBMC+isolation+have+a+moderate+effect+on+yield+but+severely+compromise+cell+viability.">Delays during PBMC isolation have a moderate effect on yield but severely compromise cell viability.</a> Clin Chem Lab Med. 60 (5), 701-706 (2022).</li><li>Fuss, I. J., Kanof, M. E., Smith, P. 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. L., Lippi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ethylenediamine+tetraacetic+acid+(EDTA)+as+in+vitro+anticoagulant+for+diagnostic+purposes.">The role of ethylenediamine tetraacetic acid (EDTA) as in vitro anticoagulant for diagnostic purposes.</a> Clin Chem Lab Med. 45 (5), 565-576 (2007).</li><li>Mohri, M., Rezapoor, H. <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+heparin,+citrate,+and+EDTA+on+plasma+biochemistry+of+sheep:+Comparison+with+serum.">Effects of heparin, citrate, and EDTA on plasma biochemistry of sheep: Comparison with serum.</a> Res Vet Sci. 86 (1), 111-114 (2009).</li><li>Edwards, K., 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=Heparin-mediated+PCR+interference+in+SARS-CoV-2+assays+and+subsequent+reversal+with+heparinase+I.">Heparin-mediated PCR interference in SARS-CoV-2 assays and subsequent reversal with heparinase I.</a> J Virol Methods. 327, 114944 (2024).</li><li>Yokota, M., Tatsumi, N., Nathalang, O., Yamada, T., Tsuda, 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+heparin+on+polymerase+chain+reaction+for+blood+white+cells.">Effects of heparin on polymerase chain reaction for blood white cells.</a> J Clin Lab Anal. 13 (3), 133-140 (1999).</li><li>Yuan, Y., 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=Efficient+long-term+cryopreservation+of+pluripotent+stem+cells+at+-80+&#160;&#176;C.">Efficient long-term cryopreservation of pluripotent stem cells at -80 &#160;&#176;C.</a> Sci Rep. 6 (1), 34476 (2016).</li></ol>

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><tbody><tr><td>1000 &amp;micro;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&lt;br/&gt; 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 &amp;micro;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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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.&lt;br/&gt; 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&lt;br/&gt; 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 &amp;micro;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' instructions (Figure 2A-F). A mean ± SD viability of PBMCs, monocytes, and lymphocytes of 94 ± 4.0%, 98 ± 1.1%, and 93 ± 5.6%, respectively, was achieved (Figure 2G). Viability measurement by Trypan Blue exclusion ...

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 ...

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

Research

JoVE Journal

Medicine

2.9K Views.  University of California San Diego. In dit protocol willen we een gemakkelijk te volgen, stapsgewijze zelfstudie bieden over het verzamelen en bewaren van perifeer bloed en mononucleaire cellen met een hoge levensvatbaarheid of PBMC's uit volbloed. PBMC's omvatten lymfocyten, dendritische cellen en monocyten. Bulk PBMC's kunnen worden verwerkt om individuele celtypen te isoleren en worden vaak gebruikt in een breed scala aan experimenten en assays, waaronder RNA-sequencing en flowcytometrie.

Video: Isolatie en cryopreservatie van zeer levensvatbare mononucleaire cellen van menselijk perifeer bloed uit volbloed: een gids voor beginners

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Developmental Biology

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Cancer Research

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 ...

Examining Micronuclei Yields in Irradiated Whole Blood Samples

The Micronucleus Assay on Cryopreserved Whole Blood

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. 
As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

Author Spotlight: Cryopreservation of Whole Blood Samples for High Throughput Analysis 

Here we present an optimized protocol for the cytokinesis-block micronucleus assay on cryopreserved whole blood samples. This optimized method of cryopreservation of whole blood for micronucleus analysis is a reliable technique for large-scale sampling and multi-center studies and can be used for other blood-related assays as well.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...

Immunology and Infection

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

High-Throughput PBMC Isolation Using Immunomagnetic Beads

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Biology

Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

Source: Mily, A., et al. <a href="https://app.jove.com/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further analysis.

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video