{"id":2159,"date":"2025-05-14T10:00:05","date_gmt":"2025-05-14T08:00:05","guid":{"rendered":"https:\/\/semmelweis.hu\/kutlab\/?page_id=2159"},"modified":"2025-10-10T11:58:06","modified_gmt":"2025-10-10T09:58:06","slug":"munkatarsak-122-32479-modszerek-sumeg","status":"publish","type":"page","link":"https:\/\/semmelweis.hu\/kutlab\/munkatarsak-122-32479-modszerek-sumeg\/","title":{"rendered":"M\u00f3dszerek-Membr\u00e1ntranszport konferencia 2025"},"content":{"rendered":"<p data-start=\"128\" data-end=\"206\"><span style=\"font-size: 18pt\"><strong data-start=\"128\" data-end=\"204\">Peripheral Blood Mononuclear Cell (PBMC) Isolation and Freezing Protocol<\/strong><\/span><\/p>\n<p data-start=\"208\" data-end=\"514\"><span style=\"font-size: 12pt\">The cellular composition of the blood sample (white blood cells [WBC], red blood cells [RBC], platelets [PLT], lymphocytes [LY], and neutrophil granulocytes [NEU]) is determined using a hematology analyzer (Sysmex). The sample is then diluted threefold with PBS (Gibco) containing 2 mM EDTA (Invitrogen).<\/span><\/p>\n<p data-start=\"516\" data-end=\"990\"><span style=\"font-size: 12pt\">White blood cells are separated from granulocytes and red blood cells by Ficoll density gradient centrifugation. The diluted sample (30\u201335 ml) is carefully layered onto 15 ml Ficoll solution (GE Healthcare, Merck Life Science) and centrifuged for 30 minutes at 400 g with the brake off. The thin PBMC layer formed above the Ficoll interface is gently collected with a pipette into a sterile tube, washed with PBS\u2013EDTA buffer, and centrifuged again for 10 minutes at 300 g.<\/span><\/p>\n<p data-start=\"992\" data-end=\"1605\"><span style=\"font-size: 12pt\">The pellets are resuspended in 20 ml PBS\u2013EDTA buffer. The total volume is measured, and 1 ml of the sample is set aside for cell counting using the Sysmex analyzer and for FACS analysis. A second cell count is then performed. The remaining cell suspension is adjusted with PBS\u2013EDTA to allow convenient dilution for freezing. The suspension is centrifuged again for 10 minutes at 300 g, the supernatant is removed, and the pellet is gently resuspended. An appropriate volume of freezing medium (50% fetal bovine serum [FBS, PAN Biotech], 40% RPMI basal medium [Gibco], 10% DMSO [Miltenyi]) is added to the cells.<\/span><\/p>\n<p data-start=\"1607\" data-end=\"1766\"><span style=\"font-size: 12pt\">The cell suspension is aliquoted into 1 ml cryovials and frozen at \u221280 \u00b0C for a maximum of 2 days, then transferred to liquid nitrogen for long-term storage.<\/span><\/p>\n<p style=\"text-align: justify\"><span style=\"font-size: 12pt\">Previously collected samples are characterized by flow cytometry after staining with surface markers CD3, CD4, CD8, CD20, CD14, and CD56.<\/span><\/p>\n<h2>\u00a0<\/h2>\n<p data-start=\"82\" data-end=\"179\"><span style=\"font-size: 24pt\"><strong data-start=\"82\" data-end=\"147\">Lymphocyte Population Identification by Flow Cytometry (FACS)<\/strong><\/span><\/p>\n<p data-start=\"82\" data-end=\"179\"><br data-start=\"147\" data-end=\"150\" \/><br \/>\n<span style=\"font-size: 18pt\"><strong data-start=\"150\" data-end=\"179\">Analysis of Dot Plot Data<\/strong><\/span><\/p>\n<p data-start=\"181\" data-end=\"378\"><span style=\"font-size: 12pt\">Within the PBMC sample, individual cell populations were distinguished based on cell size (FSC) and granularity (SSC) using flow cytometry. Three distinct cell populations were clearly separated:<\/span><\/p>\n<ul data-start=\"379\" data-end=\"785\">\n<li data-start=\"379\" data-end=\"512\">\n<p data-start=\"381\" data-end=\"512\"><span style=\"font-size: 12pt\"><strong data-start=\"381\" data-end=\"388\">PLT<\/strong> denotes platelets (thrombocytes), which are the smallest in size and lack a nucleus, resulting in the lowest granularity.<\/span><\/p>\n<\/li>\n<li data-start=\"513\" data-end=\"593\">\n<p data-start=\"515\" data-end=\"593\"><span style=\"font-size: 12pt\"><strong data-start=\"515\" data-end=\"521\">MO<\/strong> denotes monocytes, which are the largest cells and contain a nucleus.<\/span><\/p>\n<\/li>\n<li data-start=\"594\" data-end=\"785\">\n<p data-start=\"596\" data-end=\"785\"><span style=\"font-size: 12pt\"><strong data-start=\"596\" data-end=\"602\">LY<\/strong> denotes lymphocytes, the population of primary interest in this study. Lymphocytes have an average diameter of 6\u20137 \u00b5m and are of intermediate size compared to the other cell types.<\/span><\/p>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone  wp-image-2169\" src=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/PBMD-gateles-1.png\" alt=\"\" width=\"373\" height=\"343\" srcset=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/PBMD-gateles-1.png 1007w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/PBMD-gateles-1-400x368.png 400w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/PBMD-gateles-1-768x707.png 768w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/PBMD-gateles-1-753x693.png 753w\" sizes=\"auto, (max-width: 373px) 100vw, 373px\" \/><\/p>\n<h2>\u00a0<\/h2>\n<p data-start=\"96\" data-end=\"168\"><span style=\"font-size: 24pt\"><strong data-start=\"96\" data-end=\"122\">CFSE Staining Protocol<\/strong><\/span><br data-start=\"122\" data-end=\"125\" \/><br \/>\n<span style=\"font-size: 18pt\"><strong data-start=\"125\" data-end=\"168\">Theoretical Background of CFSE Staining<\/strong><\/span><\/p>\n<p data-start=\"170\" data-end=\"557\"><span style=\"font-size: 12pt\">CFSE (Invitrogen), short for <em data-start=\"199\" data-end=\"248\">carboxyfluorescein diacetate succinimidyl ester<\/em>, is a cell-permeable fluorescent dye. It readily diffuses through the cell membrane into the cytoplasm, where intracellular esterases cleave off the acetate groups. The resulting fluorescent molecule can no longer cross the membrane and becomes trapped inside the cytoplasm, providing stable cell labeling.<\/span><\/p>\n<p data-start=\"559\" data-end=\"919\"><span style=\"font-size: 12pt\">During cell division, the CFSE-containing cytoplasm is evenly distributed between daughter cells, leading to a halving of fluorescence intensity with each division cycle. By detecting the proportion of cells in which CFSE fluorescence intensity has decreased compared to the initial value, the percentage (or number) of proliferating cells can be determined.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone size-full wp-image-2168\" src=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas.png\" alt=\"\" width=\"3805\" height=\"1739\" srcset=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas.png 3805w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas-400x183.png 400w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas-1024x468.png 1024w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas-768x351.png 768w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas-1536x702.png 1536w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas-2048x936.png 2048w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-jeintenzitas-753x344.png 753w\" sizes=\"auto, (max-width: 3805px) 100vw, 3805px\" \/><\/p>\n<p style=\"text-align: justify\">\n<p>&nbsp;<\/p>\n<p data-start=\"152\" data-end=\"178\"><span style=\"font-size: 18pt\"><strong data-start=\"152\" data-end=\"178\">CFSE Staining Protocol<\/strong><\/span><\/p>\n<p data-start=\"180\" data-end=\"700\"><span style=\"font-size: 12pt\">Previously frozen PBMCs were used for the staining procedure. After thawing, cells were resuspended in 4 ml HBSS. Following homogenization, a sample was taken, and the cell suspension was centrifuged for 5 minutes at 350 g in a pre-cooled centrifuge (6 \u00b0C). Cell counts were determined from the collected sample using a Sysmex hematology analyzer. After centrifugation, the supernatant was discarded, the pellet was gently resuspended, and the cells were adjusted with PBS to a final concentration of 1 \u00d7 10\u2077 cells\/ml.<\/span><\/p>\n<p data-start=\"702\" data-end=\"1068\"><span style=\"font-size: 12pt\">The homogenized cell suspension was transferred to an empty tube for labeling. Cells were stained with CFSE at a final concentration of 1 \u00b5M. The samples were incubated in the dark for 20 minutes at room temperature, allowing the dye to diffuse across the cell membrane and enter the cytoplasm. Meanwhile, unstained control samples were measured by flow cytometry.<\/span><\/p>\n<p data-start=\"1070\" data-end=\"1497\"><span style=\"font-size: 12pt\">After incubation, 4 ml of complete RPMI medium (RPMI supplemented with 10% FBS [PAN Biotech], 1% Penicillin-Streptomycin [Merck Life Science], and 1% GlutaMAX [Gibco]) was added to the cells. The suspension was centrifuged for 5 minutes at 400 g at room temperature. The supernatant was removed, the pellet was resuspended in 4 ml complete RPMI, and the cells were incubated for an additional 20 minutes at 37 \u00b0C in the dark.<\/span><\/p>\n<p data-start=\"1499\" data-end=\"1784\"><span style=\"font-size: 12pt\">Following the second incubation, the cells were centrifuged again for 5 minutes at 400 g at room temperature and resuspended in complete RPMI medium at the concentration required for plating. Finally, CFSE-labeled cells were analyzed by flow cytometry to confirm successful staining.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p data-start=\"138\" data-end=\"202\"><span style=\"font-size: 18pt\"><strong data-start=\"138\" data-end=\"202\">Detection of Peripheral Blood Mononuclear Cell Proliferation<\/strong><\/span><\/p>\n<p data-start=\"204\" data-end=\"536\"><span style=\"font-size: 12pt\">After the predetermined incubation period, cells were counted, and lymphocytes, proliferating cells, and singlets were gated on the dot plots as described above. In CytExpert software (Beckman Coulter), the \u201chistogram\u201d display mode was selected, in which the cell count (y-axis) was plotted against CFSE signal intensity (x-axis).<\/span><\/p>\n<p data-start=\"538\" data-end=\"901\"><span style=\"font-size: 12pt\">First, an untreated control sample was used to define the P1 region, covering the x-axis from the initial CFSE peak toward decreasing signal intensities. Each sample was then evaluated based on this P1 region to determine the proportion of proliferating singlet cells showing reduced CFSE intensity, indicating the fraction of cells that had undergone division.<\/span><\/p>\n<p data-start=\"903\" data-end=\"993\"><span style=\"font-size: 12pt\">The resulting percentages were plotted and analyzed using GraphPad Prism 9.1.2 (GraphPad).<\/span><\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone  wp-image-2167\" src=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles.png\" alt=\"\" width=\"328\" height=\"333\" srcset=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles.png 1855w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles-393x400.png 393w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles-1007x1024.png 1007w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles-768x781.png 768w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles-1510x1536.png 1510w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles-753x766.png 753w\" sizes=\"auto, (max-width: 328px) 100vw, 328px\" \/> <img loading=\"lazy\" decoding=\"async\" class=\"alignnone  wp-image-2170\" src=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles_ConA-1.png\" alt=\"\" width=\"314\" height=\"320\" srcset=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles_ConA-1.png 1852w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles_ConA-1-392x400.png 392w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles_ConA-1-1003x1024.png 1003w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles_ConA-1-768x784.png 768w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles_ConA-1-1505x1536.png 1505w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/CFSE-kiertekeles_ConA-1-753x768.png 753w\" sizes=\"auto, (max-width: 314px) 100vw, 314px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p data-start=\"219\" data-end=\"310\"><span style=\"font-size: 18pt\"><strong data-start=\"219\" data-end=\"310\">Inhibition of Peripheral Blood Mononuclear Cell Proliferation by Mesenchymal Stem Cells<\/strong><\/span><\/p>\n<p data-start=\"312\" data-end=\"1620\"><span style=\"font-size: 12pt\">For each experiment, the mesenchymal stem cell (MSC) line available in the laboratory at the time\u2014previously seeded and expanded over the preceding days\u2014was used. The required number of MSCs was calculated based on the number of PBMCs to be co-cultured. The culture medium (DLH; DMEM Low Glucose (Gibco) supplemented with 5% human platelet lysate (HPL), 0.01% Gentamicin (Gibco), and Heparibene Na 25000 injection at a final concentration of 1.5 U\/mL (Ratiopharm)) was aspirated, and the cells were washed once with PBS (Gibco) to remove any residual medium. TrypLE (Gibco) was then added in a volume appropriate for the culture vessel. Detachment of MSCs was accelerated by incubation at 37 \u00b0C. Cell detachment was monitored under a light microscope. Once the cells had detached, the enzymatic reaction was stopped by adding M2 medium (500 mL HBSS (Gibco), 2% HPL, 0.01% Gentamicin, and Heparibene Na 25000 injection at a final concentration of 1.5 U\/mL). The cell suspension was transferred to a 15 mL centrifuge tube, and an aliquot was used to determine cell number by flow cytometry (FACS). The remaining cells were centrifuged at 400 g for 7 minutes at room temperature. The supernatant was discarded, the pellet was resuspended, and the cells were adjusted to the required concentration in DLH medium.<\/span><\/p>\n<p data-start=\"1622\" data-end=\"2165\"><span style=\"font-size: 12pt\">According to the experimental design, MSCs and CFSE-labeled PBMCs (as described above) were seeded into 96-well culture plates. When both cell types were added to the same well, they were pipetted in rapid succession to minimize time differences. A total volume of 200 \u00b5L medium was used per well. Con A diluted 1:10 in PBS was added at 0.8 \u00b5L per well, resulting in a final concentration of 2 \u00b5g\/mL. Cells were checked under a light microscope, then incubated at 37 \u00b0C in a humidified incubator with 5% CO\u2082 for the duration of the experiment.<\/span><\/p>\n<p data-start=\"2167\" data-end=\"2266\"><span style=\"font-size: 12pt\">After the incubation period, the cells were stained with CD markers and analyzed by flow cytometry.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone size-full wp-image-2165\" src=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese.png\" alt=\"\" width=\"4027\" height=\"2033\" srcset=\"https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese.png 4027w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese-400x202.png 400w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese-1024x517.png 1024w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese-768x388.png 768w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese-1536x775.png 1536w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese-2048x1034.png 2048w, https:\/\/semmelweis.hu\/kutlab\/files\/2025\/05\/kiserlet-felepitese-753x380.png 753w\" sizes=\"auto, (max-width: 4027px) 100vw, 4027px\" \/><\/p>\n<h3>\u00a0<\/h3>\n<p data-start=\"161\" data-end=\"192\"><span style=\"font-size: 18pt\"><strong data-start=\"161\" data-end=\"192\">CD Marker Staining Protocol<\/strong><\/span><\/p>\n<p data-start=\"194\" data-end=\"535\"><span style=\"font-size: 12pt\">For each sample, 1 \u00b5L of antibody or antibody mix was added per sample regardless of the cell number (up to 1 \u00d7 10\u2076 cells). The cells were incubated for 20 minutes at room temperature in the dark. Following incubation, samples containing fewer than 0.5 \u00d7 10\u2076 cells were diluted fivefold with PBS, and subsequently analyzed by flow cytometry.<\/span><\/p>\n<p data-start=\"537\" data-end=\"627\"><span style=\"font-size: 12pt\"><strong data-start=\"537\" data-end=\"556\">Flow cytometer:<\/strong> CytoFLEX (Beckman Coulter)<\/span><br data-start=\"583\" data-end=\"586\" \/><br \/>\n<span style=\"font-size: 12pt\"><strong data-start=\"586\" data-end=\"599\">Software:<\/strong> CytExpert (Beckman Coulter)<\/span><\/p>\n<p data-start=\"629\" data-end=\"651\"><span style=\"font-size: 12pt\"><strong data-start=\"629\" data-end=\"649\">Antibodies used:<\/strong><\/span><\/p>\n<ul data-start=\"652\" data-end=\"875\">\n<li data-start=\"652\" data-end=\"690\">\n<p data-start=\"654\" data-end=\"690\"><span style=\"font-size: 12pt\">anti-human CD4-APC\/Cy7 (BioLegend)<\/span><\/p>\n<\/li>\n<li data-start=\"691\" data-end=\"727\">\n<p data-start=\"693\" data-end=\"727\"><span style=\"font-size: 12pt\">anti-human CD8-APC (ImmunoTools)<\/span><\/p>\n<\/li>\n<li data-start=\"728\" data-end=\"766\">\n<p data-start=\"730\" data-end=\"766\"><span style=\"font-size: 12pt\">anti-human CD16-PE\/Cy7 (BioLegend)<\/span><\/p>\n<\/li>\n<li data-start=\"767\" data-end=\"804\">\n<p data-start=\"769\" data-end=\"804\"><span style=\"font-size: 12pt\">anti-human CD8-PE\/Cy7 (BioLegend)<\/span><\/p>\n<\/li>\n<li data-start=\"805\" data-end=\"839\">\n<p data-start=\"807\" data-end=\"839\"><span style=\"font-size: 12pt\">anti-human CD20-PE (BioLegend)<\/span><\/p>\n<\/li>\n<li data-start=\"840\" data-end=\"875\">\n<p data-start=\"842\" data-end=\"875\"><span style=\"font-size: 12pt\">anti-human CD56-APC (ImmunoTools)<\/span><\/p>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Peripheral Blood Mononuclear Cell (PBMC) Isolation and Freezing Protocol The cellular composition of the blood sample (white blood cells [WBC], red blood cells [RBC], platelets [PLT], lymphocytes [LY], and neutrophil granulocytes [NEU]) is determined using a hematology analyzer (Sysmex). The sample is then diluted threefold with PBS (Gibco) containing 2 mM EDTA (Invitrogen). White blood &hellip;<\/p>\n","protected":false},"author":101100,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"categories":[],"tags":[],"class_list":["post-2159","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/pages\/2159","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/users\/101100"}],"replies":[{"embeddable":true,"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/comments?post=2159"}],"version-history":[{"count":6,"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/pages\/2159\/revisions"}],"predecessor-version":[{"id":2240,"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/pages\/2159\/revisions\/2240"}],"wp:attachment":[{"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/media?parent=2159"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/categories?post=2159"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/semmelweis.hu\/kutlab\/wp-json\/wp\/v2\/tags?post=2159"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}