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Pdpn 8.1.1

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Pdpn (8.1.1.) is a monoclonal antibody reagent produced by BioLegend. It is designed for the detection of Podoplanin, a type I transmembrane glycoprotein expressed on various cell types.

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Lab products found in correlation

Ef506, by Thermo Fisher Scientific (2 mentions) Pd 1 rmp1 30, by BioLegend (2 mentions) Lag 3 c9b7w, by BioLegend (2 mentions) Tigit gigd7, by BioLegend (2 mentions) Tim 3 5d12, by BioLegend (2 mentions) Procr ebio1560, by BioLegend (2 mentions) Ionomycin, by Merck Group (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Lsr 2, by BD (2 mentions) Cd31 mec 13, by BD (2 mentions) Cd45.2 104, by BD (2 mentions) Countbright absolute counting beads, by Thermo Fisher Scientific (2 mentions) Ghostdye510 viability dye, by Cytek Biosciences (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Liberase, by Roche (2 mentions) Fitc active caspase 3 apoptosis kit, by BD (1 mentions) Brdu flow kit, by BD (1 mentions) Ki67 sola15, by Thermo Fisher Scientific (1 mentions) Cd45 30 f11, by Thermo Fisher Scientific (1 mentions) Cd21 cd35, by BD (1 mentions)

6 protocols using pdpn 8.1.1

1

Multiparameter Flow Cytometry Analysis

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Single cell suspensions were stained with antibodies against CD4 (RM4–5), CD8 (53–6.7), PD-1 (RMP1–30), Lag-3 (C9B7W), TIGIT (GIGD7), and Tim-3 (5D12), Procr (eBio1560), and Pdpn (8.1.1.) were obtained from BioLegend (San Diego, CA). Fixable viability dye eF506 (eBioscience) was used to exclude dead cells. For intra-cytoplasmic cytokine staining, cells were stimulated with (PMA) (50ng/ml, Sigma-Aldrich, MO), ionomycin (1µg/ml, Sigma-Aldrich, MO). Permeabilized cells were then stained with antibodies against IL-2, TNF-α, IFN-γ or IL-10. All data were collected on a BD LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star).
Chihara N., Madi A., Kondo T., Zhang H., Acharya N., Singer M., Nyman J., Marjanovic N.D., Kowalczyk M.S., Wang C., Kurtulus S., Law T., Etminan Y., Nevin J., Buckley C.D., Burkett P.R., Buenrostro J.D., Rozenblatt-Rosen O., Anderson A.C., Regev A, & Kuchroo V.K. (2018). Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature, 558(7710), 454-459.
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2

Isolation and Analysis of Lymphoid and Splenic Cells

Cited 1 time
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LNs, and spleens where indicated, were incubated in digestion medium (RPMI, 0.1 mg/ml DNase I (Invitrogen), 0.1–0.2 mg/ml liberase (Roche) and 0.8 mg/ml dispase (Roche)19 (link). Collected single-cell suspensions were filtered. Spleen samples underwent red blood cell lysis. For flow cytometry, cells were stained with Ghostdye510 viability dye (Tonbo Biosciences) in PBS, followed by labeling with CD45 (30-F11, Invitrogen), CD45.2 (104, BD Biosciences), PDPN (8.1.1, Biolegend), CD31 (MEC 13.3, BD Biosciences) and Ki67 (SolA15, eBioscience), Madcam-1 (MECA-89, BD), CD21/CD35 (Clone: 7G6, BD), Cpt1a (8F6AE9, Abcam), CD4 (GK1.5, Invitrogen), B220 (RA3–6B2, BD), IL-17RA (PAJ-17R, Invitrogen) and CountBright™ Absolute Counting Beads (Molecular Probes). For detection of apoptosis, FRC were stained with FITC Active Caspase-3 Apoptosis Kit (BD Biosciences), as per manufacturer’s protocol. For in vivo cell cycle analysis, mice were injected i.p. with 1 mg of bromodeoxyuridine (BrdU flow kit; BD Biosciences) 24 h before sacrifice. Data acquired with a FACS Fortessa (BD Biosciences), analyzed using FlowJo (Tree Star).
Majumder S., Amatya N., Revu S., Jawale C.V., Wu D., Rittenhouse N., Menk A., Kupul S., Du F., Raphael I., Bhattacharjee A., Siebenlist U., Hand T.W., Delgoffe G.M., Poholek A.C., Gaffen S.L., Biswas P.S, & McGeachy M.J. (2019). IL-17 metabolically reprograms activated fibroblastic reticular cells for proliferation and survival. Nature immunology, 20(5), 534-545.
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3

Isolation and Sorting of Murine Liver Stromal Cells

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Briefly, murine livers were harvested and nonparenchymal cells were isolated as previously described.54 Following isolation, cells were washed 2× with PBS and dead cells were removed using the EasySep Dead Cell Removal Kit (Stem Cell Technologies, Vancouver, Canada). Viable cells were washed 2× with PBS containing 0.5% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) and stained with antibodies against CD45 (30-F11), CD31 (390), CD146 (ME-9F1), and PDPN (8.1.1) from BioLegend (San Diego, CA). Cells were washed twice and were sorted using an aria Fusion sorter (BD Biosciences, Franklin Lakes, NJ). Enriched stromal cells were then washed 2× with PBS and subjected to single cell sequencing as described subsequently.
Burchill M.A., Finlon J.M., Goldberg A.R., Gillen A.E., Dahms P.A., McMahan R.H., Tye A., Winter A.B., Reisz J.A., Bohrnsen E., Schafer J.B., D’Alessandro A., Orlicky D.J., Kriss M.S., Rosen H.R., McCullough R.L, & Jirón Tamburini B.A. (2020). Oxidized Low-Density Lipoprotein Drives Dysfunction of the Liver Lymphatic System. Cellular and Molecular Gastroenterology and Hepatology, 11(2), 573-595.
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4

Glucose uptake and flow cytometry analysis of lung-resident immune cells

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MLNs were incubated at 37°C in digestion medium (RPMI, 0.1 mg/ml DNase I (Invitrogen), 0.1 mg/ml liberase (Roche). Collected single-cell suspensions were filtered. For flow cytometry, cells were stained with Ghostdye510 viability dye (Tonbo Biosciences) in phosphate buffered saline, followed by labeling with CD45 (clone 30-F11, Invitrogen), CD45.2 (104, BD Biosciences), PDPN (8.1.1, BioLegend), CD31 (MEC 13.3, BD Biosciences), H3K4me3 (C42D8, Cell Signaling Technology) and CountBright Absolute Counting Beads (Molecular Probes). For glucose uptake assays, 500 nM of 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (Cayman Chemicals) was injected via tail vein 30 min prior to sacrifice.
Wu D., Poholek C.H., Majumder S., Liu Q., Revu S.K., Mohib K., Rothstein D.M, & McGeachy M.J. (2021). IL-17 dependent fibroblastic reticular cell training boosts tissue protective mucosal immunity through IL-10 producing B cells. Science immunology, 6(66), eaao3669.
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5

Multiparameter Flow Cytometry Analysis

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Single cell suspensions were stained with antibodies against CD4 (RM4–5), CD8 (53–6.7), PD-1 (RMP1–30), Lag-3 (C9B7W), TIGIT (GIGD7), and Tim-3 (5D12), Procr (eBio1560), and Pdpn (8.1.1.) were obtained from BioLegend (San Diego, CA). Fixable viability dye eF506 (eBioscience) was used to exclude dead cells. For intra-cytoplasmic cytokine staining, cells were stimulated with (PMA) (50ng/ml, Sigma-Aldrich, MO), ionomycin (1µg/ml, Sigma-Aldrich, MO). Permeabilized cells were then stained with antibodies against IL-2, TNF-α, IFN-γ or IL-10. All data were collected on a BD LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star).
Chihara N., Madi A., Kondo T., Zhang H., Acharya N., Singer M., Nyman J., Marjanovic N.D., Kowalczyk M.S., Wang C., Kurtulus S., Law T., Etminan Y., Nevin J., Buckley C.D., Burkett P.R., Buenrostro J.D., Rozenblatt-Rosen O., Anderson A.C., Regev A, & Kuchroo V.K. (2018). Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature, 558(7710), 454-459.
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6

Multiparametric Immunofluorescence Analysis of Murine Tissues

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The following primary antibodies were used for FACS and IF staining with murine tissues: Ter-119 (TER-119, Biolegend), CD45 (30-F11, Biolegend), EpCAM (G8.8, Santa Cruz Biotechnology), Pdpn (8.1.1, Biolegend), CD31 (390, Biolegend), Procr (eBio1560, Invitrogen), CD90 (53–2.1, Biolegend), CD55 (RIKO-3, Biolegend), αSMA (1A4, Sigma Aldrich), Adamdec1 (Origine #TA323936), Pcolce2 (Proteintech #10607-I-AP), C3 (11H9, Abcam), Sox6 (Abcam #ab30455), ER-TR7 (Abcam #ab51824), Collagen I (Abcam #ab34710), Collagen VI (Abcam #ab6588), and Fibronectin (Abcam #ab2413). The following secondary antibodies were used: donkey anti-rabbit PE (Poly4064, Biolegend), Alexa Fluor 488-, 546-, 568-, and 647-conjugated secondary antibodies were obtained for goat anti-rabbit, goat anti-rat, goat anti-mouse, and goat anti-Syrian hamster from Life Technologies, and DyLight 488- and 649-conjugated secondary antibodies for goat anti-Syrian hamster were obtained from Biolegend. NucBlue viability dye (Invitrogen) was spiked into single-cell suspensions before flow analysis or FACS sorting.
The following probes from Advanced Cell Diagnostics were used for FISH in murine tissues: Pi16 (Mm-Pi16-C2), Grem1 (Mm-Grem1-C3), and Agt (Mm-Agt-C1).
Jasso G.J., Jaiswal A., Varma M., Laszewski T., Grauel A., Omar A., Silva N., Dranoff G., Porter J.A., Mansfield K., Cremasco V., Regev A., Xavier R.J, & Graham D.B. (2022). Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing. PLoS Biology, 20(1), e3001532.
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