Cd68 pe

Manufactured by BioLegend

Sourced in United States

CD68-PE is a laboratory reagent used for the identification and quantification of cells expressing the CD68 surface marker. CD68 is a glycoprotein that is primarily expressed on the lysosomal membrane of monocytes, macrophages, and related cell types. The PE (Phycoerythrin) fluorescent dye is conjugated to the CD68 antibody, enabling its detection and analysis through flow cytometry or other fluorescence-based techniques.

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8 protocols using cd68 pe

Phenotypic Characterization of Macrophages

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To detect macrophage markers, cells were labelled with CD68-PE and CD163-PE/CY7 human Abs (Biolegend, San Diego, California, USA). Briefly, pre-treated macrophages were digested using PBS with 2.5 mM EDTA and stopped using PBS with 0.5% BSA. The precipitate was then resuspended using 100 µl of PBS, added with 5 µl of human FcR Blocking Agent (Biolegend, San Diego, California, USA), and finally mixed and incubated for 10 min to block the Fc receptor. After washed, cells were resuspended in 300 µl of PBS with 3 µl CD163-PC7 to stain cell surface Ab for 30 min. Then, the rupture of cells membranes was induced by fixation/permeabilization for 45 min. After PBS washing, cells were resuspended in 300 µl PBS with 3 µl of CD68-PE to stain cell intracellular Ab for 30 min. The percentage of CD68/CD163 macrophage cells was quantified by using Cytoflex flow cytometer (Beckman, Brea, California, USA).

Xu D., Chen W.Q., Liang M.X., Chen X., Liu Z., Fei Y.J., Shao X.Y., Wu Y., Zhang W, & Tang J.H. (2023). Tumor-derived small extracellular vesicles promote breast cancer progression by upregulating PD-L1 expression in macrophages. Cancer Cell International, 23, 137.

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Flow Cytometry Analysis of ATMs and Kupffer Cells

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Each sample was prepared to contain 10⁶ cells. A mixture of FcBlock reagent (BD Pharmingen, San Jose, CA, USA) and fluorophore-conjugated antibodies was added to each sample. The antibodies used for the analysis of ATMs were CD45-APC Cy7 (BioLegend, San Diego, CA, USA), CD68-APC (BioLegend), CD11c-phycoerythrin (CD11c-PE, BioLegend), and CD206-FITC (BioLegend). Antibodies for liver Kupffer cell analysis included CD45-FITC (BioLegend), F4/80-APC (BioLegend), CD68-PE (BioLegend), and CD11b-PerCp CY5.5 (BioLegend). After washing with 2% FBS/PBS solution, each sample was centrifuged at 1500 rpm and transferred in a fluorescence-activated cell sorting (FACS) tube. The analysis was conducted using a FACS Calibur flow cytometer (BD Bioscience, USA). The percentage of ATMs with CD45⁺ CD68⁺, CD45⁺ CD68⁺ CD206⁺, and CD45⁺ CD68⁺ CD11c⁺ expressed and the percentage of Kupffer cells with CD45⁺ F4/80⁺, CD45⁺ F4/80⁺ CD68⁺, and CD45⁺ F4/80⁺ CD11b⁺ expressed were analyzed using FlowJo (Tree Star, Inc., Ashland, OR, USA).

Kim M., Seol M.H, & Lee B.C. (2019). The Effects of Poncirus fructus on Insulin Resistance and the Macrophage-Mediated Inflammatory Response in High Fat Diet-Induced Obese Mice. International Journal of Molecular Sciences, 20(12), 2858.

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Phagocyte Depletion and Parasite Selection

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B6.RAG-1-/- mice were treated with 200uL i.v. of either saline (Sigma; negative control) or clodronate liposomes (clodronateliposomes.org). Flow cytometric analysis was carried out to assess the depletion of phagocytic cells. Single-cell suspensions of splenocytes were prepared, , and cells enumerated 24h after in vivo treatment. After staining with Zombie Aqua (Biolegend) for live/dead discrimination, cells were stained with monoclonal antibodies using appropriate combinations of fluorochromes (CD11c-BV786, CD169-BV605, F4/80-BV421, Ly6G-PerCPCy5.5, MHCII-FITC, Ter119-PE-Cy7, CD68-PE, Ly6c-APC-Cy7, CD11b-AF647, all from Biolegend). The samples were acquired on a BD Fortessa/X20 (BD Biosciences) using Diva acquisition software (BD Biosciences). FlowJo (Tree Star) software was used to analyze the data.
To determine the role of T-, B- and phagocytic cells in the selection of virulent parasites, B6.RAG-1-/- mice were treated with saline or clodronate liposomes as described above at days -1 and 4 after i.p. infection with 10⁵ iRBCs containing a mix of Neon-Green-expressing SBP parasites and mCherry-expressing MT parasites (see “Mixed infections” section).

Brugat T., Reid A.J., Lin J., Cunningham D., Tumwine I., Kushinga G., McLaughlin S., Spence P., Böhme U., Sanders M., Conteh S., Bushell E., Metcalf T., Billker O., Duffy P.E., Newbold C., Berriman M, & Langhorne J. (2017). Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection. Nature microbiology, 2, 16276.

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Isolation and Characterization of Aorta Cells

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Single aorta cells were prepared using methods described below with modification. The isolated aorta was dissected and cut into small pieces. Aorta pieces were digested with 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type I-s, 60 U/ml DNase I, and 450 U/ml collagenase type I purchased from Sigma-Aldrich in PBS containing 20 mM HEPES at 37°C for 1 hour. Cells were incubated with propidium iodide (catalog 5135, R&D System) and stained with CD45-Pacific Blue (catalog 57-0451) and CD14 APC at 1:1,000 (catalog 17-0141) (purchased from Bioscience) and CD19 Alex Fluor 488 at 1: 1,000 (catalog 302219), TCR PerCP/Cys5.5 at 1:1,000 (catalog 109227), CD11c PE/Cy7 at 1:1,000 (catalog 117317), and CD68 PE at 1:1,000 (catalog 12-0681) (purchased from BioLegend). Data analysis was performed using FlowJo software.

Park K., Mima A., Li Q., Rask-Madsen C., He P., Mizutani K., Katagiri S., Maeda Y., Wu I.H., Khamaisi M., Preil S.R., Maddaloni E., Sørensen D., Rasmussen L.M., Huang P.L, & King G.L. (2016). Insulin decreases atherosclerosis by inducing endothelin receptor B expression. JCI Insight, 1(6), e86574.

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Immune Cell Profiling in Rat and Mouse Blood

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To measure the dynamic changes in the proportions of immune cell populations in the peripheral blood of rats, 50 μl of blood samples was taken for flow cytometry analysis. After lysing red blood cells, the remaining leukocytes were resuspended in PBS containing 2% FBS, and then were stained with HIS48-FITC (eBioscience), CD11bc-allophycocyanin (BioLegend), CD68-PE (Biolegend) or CD206-PerCP (Abcam) for 30 min at 4°C. On the other hand, mice cells were analyzed by staining with CD11b-FITC (BD Bioscience), Gr-1-PE, Ly6G-APC (BD Bioscience), Ly6C-PerCP-Cy5.5 (eBioscience), F4/80-PE (eBioscience) or CD206-PerCP (Biolegend, #141716). Acquisition of flow cytometry data from the BD FACSCantoII flow cytometer was performed using FACSDiva software (BD Biosciences). The number of events analyzed was 10,000 per sample. Analysis was performed using FlowJo software (Tree Star).

Peng K.T., Hsieh C.C., Huang T.Y., Chen P.C., Shih H.N., Lee M.S, & Chang P.J. (2017). Staphylococcus aureus biofilm elicits the expansion, activation and polarization of myeloid-derived suppressor cells in vivo and in vitro. PLoS ONE, 12(8), e0183271.

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Comprehensive Cervical Cell Phenotyping

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The phenotype of cells collected on the cervical brushes was determined using flow cytometry. Squamous epithelial cells and dead leukocytes absorbed 4′,6-diamino-2-phenylindole (DAPI). Cells that excluded DAPI were examined using directly labelled monoclonal antibodies against the following human proteins: CD45-FITC and CD16-PerCPCy5.5 from BD Biosciences (Franklin Lakes, NJ, USA), CD163-PE, CD11b-PE, CD20-PE, CD14-PerCPCy5.5, HLA-DR-PECy7, CD19-PECy7, CD27-PECy7, CCR7-PECy7, CD11b-APC (activation epitope CBRM1/5) and CD16-APC from eBioscience (San Diego, CA, USA); CD235a-PE, CD68-PE, CD66b-PE, CD103-PE, CD11c-PE, CX3CR1-PE, CCR2-PerCPCy5.5, CD3-PECy7, CCR1-APC, CD4-APC, CD33-APC and CD64-APC from Biolegend (San Diego, CA, USA). Flow cytometric data were collected on an LSRII (BD Biosciences). A minimum of 300,000 events were collected for each group of antibodies so that even a leukocyte population consisting of 0.01% of the total host-derived cells could be reliably detected. Data were analysed using FlowJo 9.6.4 (TreeStar, Ashland, OR, USA).

Hunter P.J., Sheikh S., David A.L., Peebles D.M, & Klein N. (2016). Cervical leukocytes and spontaneous preterm birth. Journal of Reproductive Immunology, 113, 42-49.

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Multiparametric flow cytometry analysis

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Bones and spleens were crushed, cells resuspended and filtered to obtain a single-cell suspension that was analyzed by flow cytometry on a FACSCanto (BD Biosciences). Cells were stained with the following antibodies: CD45-APC, CD10-BV605, CD15-BV605 and glycophorin A-PE (from BD Biosciences) and CD33-BV421, CD19-PerCPCy5.5, CD34-APCCy7, CD68-PE, CD14-BV605, CD117-PECy7, IgM-PE, CD3-PECy7, FceRI-PE and CD25-BV421 (from BioLegend, San Diego, CA, USA). Control cells were stained with matching isotype controls. Sorting of cells was performed on a FACSAria (BD Biosciences).
For intracellular staining of phosphorylated STAT5 (signal transducer and activator of transcription 5), sorted pre-B cells were fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were stored in 90% ethanol in −80 °C until analysis. Cells were washed two times in ice cold phosphate-buffered saline before resuspension in phosphate-buffered saline with 2% fetal calf serum. Cells were kept on ice and stained with antibodies against phosphorylated STAT5 (STAT5P-Alexa Flour 647 from BD) or matching isotype controls. Levels of phosphorylated STAT5 are presented as median fluorescence intensity, normalized to the median fluorescence intensity of isotype-stained cells.

Askmyr M., Ågerstam H., Lilljebjörn H., Hansen N., Karlsson C., von Palffy S., Landberg N., Högberg C., Lassen C., Rissler M., Richter J., Ehinger M., Järås M, & Fioretos T. (2014). Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells. Blood Cancer Journal, 4(12), e269-.

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Efferocytosis of Apoptotic BMSCs by BMMs

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BMSCs were stained with CellTracker Deep Red (APC⁺, Invitrogen) and exposed to UV light for 30 min to induce apoptosis. Apoptotic BMSCs (apBMSCs) were recovered for 2 h at 37°C, enumerated via trypan blue exclusion (confirming cell death), and added to BMM cultures at a 1:1 ratio for 0.5–6 h. BMMs co-cultured with apBMSCs were harvested and stained with F4/80-FITC (Abd Serotec, CI:A3-1), fixed with 1% formalin and efferocytosis was assessed via flow cytometric (FACs) analyses (BD FACSAria™ III) for double labeled cells (FITC⁺APC⁺) reflecting engulfment. Macrophages cultured alone were harvested and stained with the following macrophage-specific antibodies: F4/80 FITC (CI:A3-1, BIORAD), CD86 PE (GL-1, Biolegend), and CD206 PE (C068C2, Biolegend). Cells were fixed, permeabilized with Permeabilization Buffer (Biolegend), incubated with CD68 PE (Y1/82A, Biolegend) and assessed via FACs analyses.

Michalski M.N., Koh A.J., Weidner S., Roca H, & McCauley L.K. (2016). Modulation of Osteoblastic Cell Efferocytosis by Bone Marrow Macrophages. Journal of cellular biochemistry, 117(12), 2697-2706.

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