{"id":8525,"date":"2016-07-14T11:00:34","date_gmt":"2016-07-14T11:00:34","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/biobanking\/?p=8525"},"modified":"2016-07-14T11:00:34","modified_gmt":"2016-07-14T11:00:34","slug":"comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/biobanking\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\/","title":{"rendered":"Comparing Three Isolation Techniques for Human Peripheral Blood Mononuclear Cells"},"content":{"rendered":"<p><img loading=\"lazy\" decoding=\"async\" src=\"http:\/\/admin.acceleratingscience.com\/biobanking\/wp-content\/uploads\/sites\/6\/2016\/07\/shutterstock_143897611.jpg\" style=\"float: left\" alt=\"Test tubes of blood being placed in a centrifuge. Image: angellodeco\/Shutterstock.com\" width=\"330\" height=\"215\" \/>Immune responses can be evaluated using human peripheral blood mononuclear cells (PBMCs). PBMCs are made up of lymphocytes (B cells, T cells and NK cells), monocytes and dendritic cells. &nbsp;Density centrifugation (Ficoll-Paque) is the most typical method to isolate PBMCs. However, this methodology may result in&nbsp;basophils not being present in&nbsp;a PBMC culture, as well as contamination with&nbsp;other granulocytes and red blood cells. Grievink et al. (2016) evaluated and compared three PBMC isolation techniques: the&nbsp;Ficoll approach, isolation by cell preparation&nbsp;tubes (CPTs) and isolation by SepMate tubes with freshly collected blood.<sup>1<\/sup> They&nbsp;described cell&nbsp;recovery, viability and functionality to assist researchers in&nbsp;selecting the most appropriate method of PBMC&nbsp;isolation for their specific downstream applications.<\/p>\n<p>The investigators collected venous blood from three volunteers into&nbsp;sodium-heparin tubes or CPTs containing&nbsp;sodium heparin. They performed each of the three different separation techniques within one hour of collection. The Ficoll approach resulted in the lowest cell recovery with an average of&nbsp;&nbsp;6 x 10<sup>5&nbsp;<\/sup>cells\/ml of whole&nbsp;blood. The&nbsp;SepMate tubes and&nbsp;CPTs resulted in cell recovery of&nbsp;8 x 10<sup>5&nbsp;<\/sup>cells\/ml and 13 x 10<sup>5&nbsp;<\/sup>cells\/ml of whole blood, respectively. Assessing for&nbsp;PBMC fractions, the Ficoll approach again produced the lowest cell recovery. However, when investigating cell viability using&nbsp;trypan blue exclusion, all three techniques demonstrated 100% viability.<\/p>\n<p>Microscopic examination showed that there was higher&nbsp;erythrocyte contamination for CPT-isolated populations&nbsp;compared to Ficoll- and SepMate-isolated populations. The researchers then used flow cytometry to&nbsp;assess the composition of the isolated PBMC populations,&nbsp;monocytes (CD14), T cells (CD3), B cells (CD20) and NK cells&nbsp;(CD56). Although they found variation between the three subjects, there were only minor differences between&nbsp;isolation techniques.<\/p>\n<p>Finally, the investigators quantified&nbsp;the&nbsp;response of PBMCs to a super antigen stimulation&nbsp;by their release of IFN&gamma;,&nbsp;IL-1&beta;, IL-6, IL-8 and TNF&alpha;.&nbsp;Ficoll-isolated PBMCs secreted&nbsp;lower levels of IFN&gamma; than those prepared from&nbsp;CPT- or SepMate-isolated PBMCs. Furthermore,&nbsp;IL-1&beta;, IL-6, IL-8 and TNF&alpha; release was&nbsp;lower for&nbsp;Ficoll-isolated PBMCs compared to CPT- and SepMate-isolated&nbsp;PBMCs.<\/p>\n<p>Grievink et al. suggest that&nbsp;PBMC isolation by CPTs and SepMate tubes is straightforward&nbsp;and requires limited sample handling compared to&nbsp;Ficoll isolation, which they found was more laborious but more cost-effective. Overall,&nbsp;SepMate&nbsp;and CPT result in a higher recovery than Ficoll isolation. F<span class=\"thread\">or CPT-isolated populations<\/span>, there is significantly greater erythrocyte&nbsp;contamination.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Reference<\/strong><\/p>\n<p>Grievink, H.W., et al. (2016) &#8220;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/27104742\" target=\"_blank\">Comparison of three isolation techniques for human peripheral blood mononuclear cells:&nbsp;Cell recovery and viability, population composition,&nbsp;and cell functionality<\/a>,&#8221; Biopreservation and Biobanking&nbsp;<span>[Epub ahead of print].<\/span><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Immune responses can be evaluated using human peripheral blood mononuclear cells (PBMCs). PBMCs are made up of lymphocytes (B cells, T cells and NK cells), monocytes and dendritic cells. &nbsp;Density centrifugation (Ficoll-Paque) is the most typical method to isolate PBMCs. However, this methodology may result in&nbsp;basophils not being present in&nbsp;a PBMC culture, as well as<\/p>\n","protected":false},"author":7,"featured_media":8524,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[4],"tags":[23,296],"division":[],"class_list":{"0":"post-8525","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-collection","8":"tag-blood","9":"tag-mononuclear-cells","10":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Three Isolation Methods for Human Peripheral Blood Mononuclear Cells<\/title>\n<meta name=\"description\" content=\"Grievink et al. compared three peripheral blood mononuclear cell (PBMC) isolation techniques. 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SepMate and CPT resulted in a higher recovery than Ficoll isolation.\" \/>\n<meta property=\"og:url\" content=\"https:\/\/www.thermofisher.com\/blog\/biobanking\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\/\" \/>\n<meta property=\"og:site_name\" content=\"Inside Biobanking\" \/>\n<meta property=\"article:publisher\" content=\"https:\/\/www.facebook.com\/thermofisher\" \/>\n<meta property=\"article:published_time\" content=\"2016-07-14T11:00:34+00:00\" \/>\n<meta property=\"og:image\" content=\"https:\/\/admin.acceleratingscience.com\/biobanking\/wp-content\/uploads\/sites\/6\/2016\/07\/shutterstock_143897611.jpg\" \/>\n\t<meta property=\"og:image:width\" content=\"500\" \/>\n\t<meta property=\"og:image:height\" content=\"326\" \/>\n\t<meta property=\"og:image:type\" content=\"image\/jpeg\" \/>\n<meta name=\"author\" content=\"Miriam Pollak\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:creator\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:site\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"Miriam Pollak\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"2 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\\\/\\\/schema.org\",\"@graph\":[{\"@type\":\"Article\",\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/#article\",\"isPartOf\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/\"},\"author\":{\"name\":\"Miriam Pollak\",\"@id\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/biobanking\\\/#\\\/schema\\\/person\\\/4059196ff73b52bc1f07844c5f4cc833\"},\"headline\":\"Comparing Three Isolation Techniques for Human Peripheral Blood Mononuclear Cells\",\"datePublished\":\"2016-07-14T11:00:34+00:00\",\"mainEntityOfPage\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/\"},\"wordCount\":483,\"commentCount\":0,\"image\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/#primaryimage\"},\"thumbnailUrl\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/biobanking\\\/wp-content\\\/uploads\\\/sites\\\/6\\\/2016\\\/07\\\/shutterstock_143897611.jpg\",\"keywords\":[\"blood\",\"mononuclear cells\"],\"articleSection\":[\"Collection\"],\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"CommentAction\",\"name\":\"Comment\",\"target\":[\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/#respond\"]}]},{\"@type\":\"WebPage\",\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/\",\"url\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/\",\"name\":\"Three Isolation Methods for Human Peripheral Blood Mononuclear Cells\",\"isPartOf\":{\"@id\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/biobanking\\\/#website\"},\"primaryImageOfPage\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/#primaryimage\"},\"image\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/biobanking\\\/comparing-three-isolation-techniques-for-human-peripheral-blood-mononuclear-cells\\\/#primaryimage\"},\"thumbnailUrl\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/biobanking\\\/wp-content\\\/uploads\\\/sites\\\/6\\\/2016\\\/07\\\/shutterstock_143897611.jpg\",\"datePublished\":\"2016-07-14T11:00:34+00:00\",\"author\":{\"@id\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/biobanking\\\/#\\\/schema\\\/person\\\/4059196ff73b52bc1f07844c5f4cc833\"},\"description\":\"Grievink et al. compared three peripheral blood mononuclear cell (PBMC) isolation techniques. 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