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Cd62l apc

Manufactured by Miltenyi Biotec
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CD62L-APC is a fluorochrome-conjugated antibody that binds to the CD62L (L-selectin) antigen on the surface of cells. CD62L is a cell adhesion molecule involved in the trafficking of lymphocytes. The APC (Allophycocyanin) fluorochrome allows for the detection and analysis of CD62L-expressing cells using flow cytometry.

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Lab products found in correlation

Cd3 fitc, by Miltenyi Biotec (2 mentions) Cd45ra pe, by BD (2 mentions) Facsverse, by Merck Group (2 mentions) Slc29a2 polyclonal antibody, by Thermo Fisher Scientific (2 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions) Cd105 sn6, by Thermo Fisher Scientific (1 mentions) Cd8 alexa fluor 700, by Beckman Coulter (1 mentions) Cd4 pacific blue, by Beckman Coulter (1 mentions) Cd3 krome orange, by Beckman Coulter (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) Fc block, by BD (1 mentions) Cd31 pe, by Miltenyi Biotec (1 mentions) Cd14 fitc, by Miltenyi Biotec (1 mentions) Kaluza software version 1, by Beckman Coulter (1 mentions) Cd3 fitc, by BD (1 mentions) Cd3 efluor 450, by BD (1 mentions) Foxp3 apc, by Miltenyi Biotec (1 mentions) Foxp3 pe, by Miltenyi Biotec (1 mentions) Cd45ra pe, by Miltenyi Biotec (1 mentions) Cd4 fitc, by R&D Systems (1 mentions)

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CD62L (3 citations)

7 protocols using cd62l apc

1

Flow Cytometric Analysis of HBEC Activation

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For flow cytometric analysis of HBEC, cells were incubated in the presence of fluorochrome-conjugated mAbs against CD105 (SN6), VCAM-1 (STA), B7-1/CD80 (2D10.4), B7-2/CD86 (IT2.2), CD40 (5C3), HLA-DR/MHC II (LN3) and CD275 (MIH12) (all from eBioscience), ICAM-1 (5.6E; Beckman Coulter) and β2-microglobulin/MHC I (TÜ99; BD Biosciences) as per manufacturer’s instructions. Flow cytometric analysis was performed on the FC 500 flow cytometer. For flow multi-colour cytometric analysis of the activation status of donor T cells, cells were incubated with the following antibody cocktail: CD69-FITC (FN50), CD40L-PE (CD154, 24-31) (eBioscience), CD8-Alexa Fluor 700, CD4-Pacific Blue, CD45RA-Alexa Fluor 750, CD3-Krome Orange (Beckman Coulter) and CD62L-APC (Miltenyi Biotec). Cells were washed once prior to flow cytometry using a Gallios™ Flow Cytometer (Beckman Coulter). Naive and memory T-cell populations were distinguished by CD45RA and CD62L expression, as described previously (37 (link)). For EMP phenotyping, HBEC supernatant following stimulation was incubated with mAb and diluted 1:1 in PBS prior to flow cytometric analysis on the FC 500 flow cytometer. EMP production per 100,000 HBEC was determined by triplicate supernatant analysis. The supernatant was collected from 400,000 cultured HBEC per well following 18 h stimulation.
Wheway J., Latham S.L., Combes V, & Grau G.E. (2014). Endothelial microparticles interact with and support the proliferation of T cells. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3378-3387.
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2

Profiling Monocyte Surface Markers

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To study the surface cell markers on monocytes (CD14 +), PBMCs from the 10 patients used for single-cell analysis and 10 HDs were defrosted and washed once with PBS. After blocking for non-specific binding with Fc block (BD Pharmingen) for 5 min on ice, cells were incubated for 20 min on ice using staining buffer (PBS with 4% fetal bovine serum and 0.4% EDTA). Antibodies used included the following: CD14-FitC (Miltenyi Biotec), CD85-PEvio770 (Miltenyi Biotec), CD172a-APC (Miltenyi Biotec), CD97-PEvio770 (Miltenyi Biotec), CD31-PE (Miltenyi Biotec), CD366-PEvio615 (Miltenyi Biotec), CD62L-APC (Miltenyi Biotec), CD58-PE (Miltenyi Biotec), CD191-PEvio770 (Miltenyi Biotec), CD52-PEvio615 (Miltenyi Biotec), CD48-APC (Miltenyi Biotec). Cells were analyzed in a BD FACSCanto-II flow cytometer.
Godoy-Tena G., Barmada A., Morante-Palacios O., de la Calle-Fabregat C., Martins-Ferreira R., Ferreté-Bonastre A.G., Ciudad L., Ruiz-Sanmartín A., Martínez-Gallo M., Ferrer R., Ruiz-Rodriguez J.C., Rodríguez-Ubreva J., Vento-Tormo R, & Ballestar E. (2022). Epigenetic and transcriptomic reprogramming in monocytes of severe COVID-19 patients reflects alterations in myeloid differentiation and the influence of inflammatory cytokines. Genome Medicine, 14, 134.
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3

PBMC Phenotyping and hENT2 Expression

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PBMC samples were seeded (1 × 104 cells/well) in complete medium with or without the indicated concentrations of compounds. After 72 h incubation, cells were washed with PBS and stained with CD45RA PE (BD-Biosciences), CD62L APC (Miltenyi Biotec), CD3 FITC (Miltenyi Biotec) at room temperature for 20 min. Data collection was done on FACSVerse and dead cells were excluded from the analysis, based on the use of PI (Sigma-Aldrich).
For the evaluation of hENT2 protein expression, cell lines or AML primary cells were incubated with a SLC29A2 polyclonal antibody (Thermo fisher Scientific) using a dilution of 1:25 for 20 min at room temperature after fixation and permeabilization. The secondary antibody (Cell signaling Technology USA, Danvers, MA, USA) was used with a dilution of 1:200 for 20 min. Results were expressed as ΔGmean, i.e., the net mean fluorescence intensity (MFI) difference between the cells stained with primary and secondary antibodies and cells stained with the secondary antibody only, in order to eliminate the background positivity. All data were processed with Kaluza software version 1.2 (Beckman Coulter).
Gaidano V., Houshmand M., Vitale N., Carrà G., Morotti A., Tenace V., Rapelli S., Sainas S., Pippione A.C., Giorgis M., Boschi D., Lolli M.L., Cilloni D., Cignetti A., Saglio G, & Circosta P. (2021). The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia. Cancers, 13(5), 1003.
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4

Multicolor Flow Cytometry Analysis of Immune Cells

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Cells were stained with CD3 FITC (Cat# 11-0039), CD3 eFluor 450 (Cat# 48-0038), CD4 PE-Cyanine7 (Cat# 25-0049), CD4 PerCP-Cyanine5.5 (Cat# 560650, BD Pharmingen), Foxp3 APC (Cat# 17-4777), Foxp3 PE (Cat# 12-4776), CD45RA PE (Cat# 130-092-248, Miltenyi), CD45RA APC (Cat# 130-092-249, Miltenyi), CD62L APC (Cat# 17-0629), CD25 PE (Cat# 130-091-024, Miltenyi), CD25 APC-H7 (Cat# 560225, BD Pharmingen), CD8 PE-Cyanine7 (Cat# 25-0088), Perforin PE (Cat# 12-9994), granzyme B PE (Cat# 12-8899), CCR8 APC (Cat# FAB1429A, R&D System), CD4 FITC (Cat# 11-0049), CD45 APC (Cat# 17-9459), CD14 PE (Cat# 12-0149), CD45RO PE (Cat# 12-0457), CD45RO eFluor 450 (Cat# 48-0457), CD127 APC (Cat# 17-1278). PITPNM3 antibody (Cat# NBP1-31070, Novus) was labeled with APC by Abcam APC Conjugation Kit (ab201807, Abcam) according to the manufacturer's instructions. For the intracellular stain, cells were pretreated with Intracellular Fixation and Permeabilization kit (Cat# 88-8824) according to the manufacturer's instructions. All the reagents were from eBioscience unless indicated otherwise. Cells were subsequently analyzed by multicolor flow cytometry (Gallios, Beckman Coulter, China).
Su S., Liao J., Liu J., Huang D., He C., Chen F., Yang L., Wu W., Chen J., Lin L., Zeng Y., Ouyang N., Cui X., Yao H., Su F., Huang J.D., Lieberman J., Liu Q, & Song E. (2017). Blocking the recruitment of naive CD4+ T cells reverses immunosuppression in breast cancer. Cell Research, 27(4), 461-482.
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5

T cell Profiling and Exhaustion Analysis

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T cell sub-population profiling studies on days 14 and 21 were analyzed using CD3-PC7 (Beckman Coulter, 6607100), CD4-APC-A700 (Beckman Coulter, B10824), CD8-PC5.5 (Beckman Coulter, B21205), EGFR-A488 (R&D Systems, FAB10951G), CD45RA-ECD (Beckman Coulter, IM2711U), CD45RO-PE (Miltenyi Biotech,130-113-559), CD27-APC-A750 (Beckman Coulter, B12701), and CD62L-APC (Miltenyi Biotech, 130-113-617) antibodies by Cytoflex Flow Cytometer analysis. Exhaustion profiling studies on days 14 and 21 were analyzed using CD3-PC7 (Beckman Coulter, 6607100), CD4-APC-A700 (Beckman Coulter, B10824), CD8-PC5.5 (Beckman Coulter, B21205), EGFR-A488 (R&D Systems, FAB10951G), CD279 (PD1)-PE (Miltenyi Biotech, 130-117-384), CD366 (TIM3)—APC (Invitrogen, Waltham, MA, USA, 17-3109-42), and CD223 (LAG-3)—APC-eFluor 780 (Invitrogen, 47-2239-42) antibodies by Cytoflex Flow Cytometer analysis.
Gulden G., Sert B., Teymur T., Ay Y., Tiryaki N.N., Mishra A.K., Ovali E., Tarhan N, & Tastan C. (2023). CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies. Vaccines, 11(2), 313.
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6

CD62L-based T Cell Sorting Protocol

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Activated T cells were labeled with cleavable CD62L-APC (clone REAL163), bound to paramagnetic anti-APC microbeads, and separated via magnetic-activated cell separation into CD62L- and CD62L+ cells according to the manufacturer’s instructions of the Anti-APC Microbeads Kit (Miltenyi Biotec, Germany). To release the CD62L-APC antibody for transduction experiments, it was enzymatically cleaved using the REAlease® Support Kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. CD62L expression on separated cells was determined by flow cytometry detecting CD62L-APC labeling or additional staining with CD62L-PE-Vio770 (clone 145/15). All antibodies were from Miltenyi Biotec (Bergisch Gladbach, Germany).
Kapitza L., Ho N., Kerzel T., Frank A.M., Thalheimer F.B., Jamali A., Schaser T., Buchholz C.J, & Hartmann J. (2023). CD62L as target receptor for specific gene delivery into less differentiated human T lymphocytes. Frontiers in Immunology, 14, 1183698.
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7

Immunophenotyping of Lymphocyte Subsets

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PBMC samples were seeded (1 × 10 4 cells/well) in complete medium with or without the indicated concentrations of compounds. After 72 hours incubation, cells were washed with PBS and stained with CD45RA PE (BD Biosciences), CD62L APC (Miltenyi Biotec), CD3 FITC (Miltenyi Biotec) at room temperature for 20 minutes. Data collection was done on FACSVerse and dead cells were excluded from the analysis, based on the use of PI (Sigma-Aldrich, Milan, Italy). Cells lines or AML primary cells were incubated with a SLC29A2 polyclonal antibody (Thermo fisher Scientific) using a dilution of 1:25 for 20 minutes at room temperature after fixation and permeabilization. The secondary antibody (Cell signaling Technology USA, Danvers, MA) was used with a dilution of 1:200 for 20 min. All data were processed with Kaluza software version 1.2.
Gaidano V., Houshmand M., Vitale N., Carrà G., Morotti A., Tenace V., Rapelli S., Sainas S., Pippione A.C., Giorgis M., Boschi D., Lolli M.L., Cignetti A., Saglio G., & Circosta P. (2020). The Synergism Between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.
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