A functional grade purified antibody for anti-mouse CD62L (anti L-selectin) was obtained from eBiosciences (clone MEL-14). Two doses of 100 µg each were given intravenously to lean and HFD fed mice on day -1 and the day 0 (day of IRI). Rat IgG2a isotype control was used in similar doses (eBiosciences).
Mel 14
Manufactured by Thermo Fisher Scientific
The MEL-14 is a laboratory instrument designed for cell separation and analysis. It utilizes magnetic cell separation technology to isolate specific cell populations from complex samples.
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Lab products found in correlation
Rat igg2a isotype control, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cxcr3 173, by Thermo Fisher Scientific (1 mentions)
Jes6 5h4, by Thermo Fisher Scientific (1 mentions)
Af6 88, by Thermo Fisher Scientific (1 mentions)
Anti cd11a, by BD (1 mentions)
Lsr 2 green, by BD (1 mentions)
Klrg1, by BD (1 mentions)
His51, by Thermo Fisher Scientific (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Detoxi gel endotoxin removing gel, by Thermo Fisher Scientific (1 mentions)
Cd49d r1 2, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
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Anti f4 80 bm8, by BioLegend (1 mentions)
Anti cd4 rm4 5, by BD (1 mentions)
Anti cd3 17a2, by BioLegend (1 mentions)
Anti cd25 pc61, by BioLegend (1 mentions)
Anti gr 1 rb6 8c5, by BD (1 mentions)
9 protocols using mel 14
1
Anti-CD62L Antibody Pretreatment in IRI
2
Multiparametric Flow Cytometry Protocol
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Single-cell suspensions were stained with antibodies or H60 tetramers (LTFNYRNL/H-2Kb) at 4 °C for 30 min in staining buffer (1 × PBS containing 0.1% bovine serum albumin and 0.1% sodium azide). Intracellular staining was performed after cell fixation and permeabilization22 (link). Cells were analysed using a FACSCalibur (BD Pharmingen, San Diego, CA, USA) or LSRII-Green (BD Pharmingen) and data were analysed using the FlowJo software (Tree Star, Ashland, OR, USA). Antibodies used for flow cytometric analysis were as follows. Fluorescent-dye-conjugated antibodies against CD8 (1:1,000; 53–6.7), CD40 (1:500; 1C10), CD44 (1:2,000; IM7), CD45.1 (1:1,000; A20), CD62L (1:2,000; MEL-14), CD80 (1:1,000; 16-10A1), CD86 (1:1,000; GL1), Thy1.1 (1:1,000; HIS51), CD122 (1:1,000; TM-b1), CD127 (1:1,000; A7R34), H-2Kb (1:500; AF6-88.5), mIgG (1:600; M1-14D12), granzyme B (1:4,000; 16G6), IFN-γ (1:2,000; XMG1.2), IL-2 (1:1,000; JES6-5H4) and CXCR3 (1:1,000; CXCR3-173) were all purchased from eBioscience. Anti-CD11a (1:1,000; 2D7), -PD-1 (1:1,000; 29 F.1A12) and -KLRG1 (1:1,000; 2F1) antibodies were purchased from BD Pharmingen. Anti-CD69 antibody (1:1,000; H1.2F3) was purchased from BioLegend (San Diego, CA, USA), and anti-Blimp-1 antibody (1:500; N-20) was from Santa Cruz Biotechnology (Dallas, TX, USA).
3
Characterization of DNA Vaccines for Allergy
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DNA vaccines (pVAX1-OVA and pVAX1-Der-p1) were produced previously (82 (link)) and prepared by EndoFree Plasmid Maxi Kit (Qiagen). His-tagged recombinant Der p 1 protein was prepared by Ni-NTA agarose (Qiagen) and Detoxi-Gel Endotoxin Removing Gel (Thermo). Endotoxin of each vaccine was below 10 EU/mg. All reagents were from Sigma-Aldrich unless specified otherwise. Magnetic CD4+ T cell purification kit was purchased from R&D System. LTβR-Ig protein was a kind gift from Dr. Yangxin Fu (University of Chicago) and Dr. Mingzhao Zhu (Institute of Biophysics, Chinese Academy of Science). Lentiviral systems of S1p1 overexpression, CXCR3, and VLA-4 knockdown were gifts from Dr. Ying Wan (Third Military Medical University, Chongqing, China). Antibodies for CCR5 (7A4), CCR7 (4B12), CD44 (IM7), CD62L (MEL-14), CD4 (GK1.5), CD25 (PC61.5), CD69 (H1.IF3), DO11.10 TCR (KJ1-26), IL-35/IL-12p35 (4D10p35), B220 (RA3-6B2), and MHC-II (M5/114.15.2), as well as all ELISA kits, were purchased from eBioscience. Antibodies for CCR3 (TG14), CXCR3 (CXCR3-173), CCR6 (29-2L17), CCR9 (9B6), CD11a (M17/4), and CD49d (R1-2) were from Biolegend. Antibodies for S1p1 (H-60), CCR8 (c-17), and Ki67 (M-19) were from Santa Cruz. Antibody for IL35/EBI3 was from R&D System.
4
Multi-Dimensional Flow Cytometry Analysis
5
Multiparameter Flow Cytometry Analysis of Immune Cells
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To analyze immune cells by flow cytometry, tumors or spleens of mice after various treatments were collected and stained according to the manufacturer’s protocols. In brief, cells from tumors were blocked with CD16/32 antibody (BioLegend, catalog no. 103102) and then stained with antibodies against CD45 (BioLegend, catalog no. 103108, clone 30-F11), CD11b (BioLegend, catalog no.101208, clone M1/70), CD206 (BioLegend, catalog no. 141716, clone C068C2), F4/80 (BioLegend, catalog no. 123116, clone BM8), CD80 (BioLegend, catalog no. 104722, clone16-10A1), CD3 (clone 17A2, catalog no. 100204), CD8a (clone 53-6.7, catalog no. 100712), and CD4 (clone GK1.5, catalog no. 100432). To analyze memory T cells, spleens of mice were stained with anti-CD3–FITC (fluorescein isothiocyanate), anti-CD8–APC (allophycocyanin), anti-CD62L–PerCP (peridinin chlorophyll protein)–Cy5.5 (eBioscience, clone MEL-14, catalog no. 17-0621), and anti-CD44–PE (phycoerythrin) (eBioscience, clone IM7, catalog no. 12-0441) antibodies to identify TCM (central memory T) cells (CD3+CD8+CD62L+CD44+) and TEM cells (CD3+CD8+CD62L−CD44+).
6
Isolation of Primary Polymorphonuclear Cells
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Whole blood was collected from one healthy volunteer using Vacuette Safety G21 and lithium heparin‐coated Vacuette tubes (both Greiner Bio One International GmbH). Isolation of primary PMNs was executed as described by Oh et al. (2008 (link)). Briefly, 5 ml of whole blood was carefully overlaid onto 5 ml Lympholyte‐poly cell separation media (Cedarlane) and centrifuged at 2,000 revolutions per minute and 20°C for 35 min. While the upper three of the six resulting layers were disposed, the layers containing PMNs and separation media were collected and washed with Hank's Balanced Salt solution (HBSS; Gibco™). After centrifugation for 10 min, supernatants were removed and the pellet was resuspended with 2 ml Red Blood Cell Lysis buffer (Santa Cruz). Subsequently, cells were centrifuged for 5 min and the lysing process was repeated. The supernatants were discarded, and the pellet was resuspended with 10 ml HBSS and centrifuged for 5 min, followed by resuspension of the PMNs pellet with HBSS containing Ca2+ and Mg2+. After confirmation of PMNs purity by flow cytometry analysis of surface markers CD16 (eBioCB16 (CB16); FITC, eBioscience) and CD62L (MEL‐14; FITC, eBioscience™), cells were immediately used for experiments.
7
Orthotopic Pancreatic Tumor Dissection
8
Isolation and Analysis of Immune Cells
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Single-cell suspensions of spleen, mesenteric lymph nodes or a pool of subcutaneous lymph nodes (Lnn. mandibularis, Lnn. cervicales superficiales, Lnn. axillares et cubiti, Lnn. inguinales superficiales, and Lnn. subiliaci) were prepared using 70 μm cell strainers (BD Biosciences). Bone marrow cells were harvested from femurs and tibias by flushing the cavities of intact bones with flow cytometry buffer (Hank's buffered salt solution, supplemented with 5% fetal calf serum and 10 mM HEPES), followed by filtration through 70 μm cell strainers. Single-cell suspensions from the spleen and bone marrow were subjected to red blood cell lysis (erythrocyte lysis buffer, Qiagen). Monoclonal antibodies to CD4 (RM4-5, GK1.5), CD8 (53–6.7), CD19 (1D3), CD25 (PC61, 7D4), CD69 (H1.2F3), CXCR4 (2B11), CCR7 (4B12), CCR9 (CW-1.2), CD62L (MEL-14), and CD44 (IM 7) and PE- and PE-Cy7-conjugated streptavidin were obtained from ThermoFisher (eBioscience) or BD Biosciences. Before flow cytometry, for some experiments, CD4+ or CD25+ cells were enriched from single-cell suspensions using biotinylated antibodies directed against CD4 or CD25, respectively, streptavidin-conjugated microbeads, and the AutoMACS magnetic cell separation system (Miltenyi Biotec). Samples were analyzed on an LSRII or LSR Fortessa system (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).
9
Leukocyte Adhesion Assay Protocol
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MLECs from control and Podxl-deficient mice were grown to confluence on 96-well plates and treated with 50 ng/mL TNFα plus 100 ng/mL IL-1β for 6 h. ICAM-2 was blocked by incubation with 5 µg/mL anti-ICAM-2 for 30 min. PU5-1.8 cells were stained with 2.5 µM calcein-AM for 30 min and, after washing, L-selectin was blocked by incubation with 5 µg/mL anti-L-selectin (MEL-14, eBioscience) for 30 min. Then, 5x10 4 PU5-1.8 cells were plated onto monolayers of MLECs in triplicate and incubated for 30 min. Non-adherent cells were removed by washes with PBS and adherent cells were quantified using a Varioskan plate reader.
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