Cd49d

Manufactured by BioLegend

Sourced in United States

CD49d is a cell surface protein that functions as a subunit of the VLA-4 integrin. It is expressed on various cell types, including lymphocytes, monocytes, and eosinophils.

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Lab products found in correlation

F4 80, by BioLegend (3 mentions) Pd1 clone 29f 1a12, by BioLegend (3 mentions) Cd62l, by BioLegend (3 mentions) Intracellular fixation permeabilization buffer set, by Thermo Fisher Scientific (2 mentions) Cd16 32 antibody, by BioLegend (2 mentions) Cd103 clone 2e7, by BioLegend (2 mentions) Thy1.1 clone ox 7, by BioLegend (2 mentions) Clone fjk 16s, by Thermo Fisher Scientific (2 mentions) 4 1bb clone 17b5, by BioLegend (2 mentions) Clone uc10 4b9, by BioLegend (2 mentions) Macsquant analyzer, by Miltenyi Biotec (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Cd11c, by Thermo Fisher Scientific (1 mentions) Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions) Facsdiva, by BD (1 mentions) Cd11b, by BioLegend (1 mentions) Human ab serum, by Atlanta Biologicals (1 mentions) Human blood type ab plasma, by LGC (1 mentions) 255 sc cf, by R&D Systems (1 mentions) Hoechst 33342, by Merck Group (1 mentions)

13 protocols using cd49d

Multicolor Flow Cytometry of Immune Cells

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Purified cell populations from spleens, colons and mesenteric lymph nodes were
stained with a cocktail of leucocyte cell surface markers including the
following anti-mouse monoclonal antibodies: CD11b (Brilliant Violet 421 #101235
[1:1000]; PE #557397 [1:500]), CD49d (FITC #103605 [1:1000]), Gr1 (PerCP-Cy5.5
#552093 [1:1000]), Ly6C (PerCP #128028 [1:1000]), F4/80 (Alexa Fluor 700 #123130
[1:1000]), Ly6G (APC/Cy7 #127624 [1:500]), CD45 (V500 #561487 [1:500]) (all
Biolegend, San Diego, CA) and CD11c (APC #17-0114-82 [1:1000]) (eBioscience, San
Diego, CA). CountBright absolute counting beads (Molecular Probes, Eugene, OR)
were used to determine total cell number per sample, and propidium iodide was
used for viability. Data collection was performed on a BD LSR II Flow Cytometer
and data analysis performed using FACSDiva (both BD Biosciences, Franklin Lakes,
NJ). On the basis of forward and side scatter, propidium iodide staining, and
staining on unutilized wave lengths, the following events were eliminated:
debris, red blood cells, aggregates, dead cells and autofluorescence.

Däbritz J., Judd L.M., Chalinor H.V., Menheniott T.R, & Giraud A.S. (2016). Altered gp130 signalling ameliorates experimental colitis via myeloid cell-specific STAT3 activation and myeloid-derived suppressor cells. Scientific Reports, 6, 20584.

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Erythroid Differentiation of CD34+ Cells

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Erythroid differentiation of CD34⁺ cells was performed using a 3-phase protocol^{42 ,43}. Phase 1 (days 1–7): IMDM (Thermo Fisher Scientific, 12440061) with 2% human blood type AB plasma (SeraCare, 1810-0001), 3% human AB serum (Atlanta Biologicals, S40110) 1% penicillin/streptomycin (Thermo Fisher Scientific, 15070063), 3 units/mL heparin (Sagent Pharmaceuticals, NDC# 25021-401-02), 3 units/mL EPO (Amgen, EPOGEN NDC # 55513-144-01), 200 μg/mL holo-transferrin (Millipore Sigma, T0665, 10 ng/mL human SCF (R&D systems, 255-SC/CF), and 1 ng/mL human interleukin IL-3 (R&D systems, 203-IL/CF). Phase 2 (days 8–14): Phase 1 medium without IL-3. Phase 3 (days 15–21): Phase 2 medium without SCF and with holo-transferrin concentration increased to 1 mg/mL. Cells were maintained daily at a density of 0.1 x 10⁶/mL (phase 1), 0.2 x 10⁶/mL (phase 2) and 1.0 x 10⁶/mL (phase 3)
Erythroblast maturation was monitored by immuno-flow cytometry for the cell surface markers CD235a (BD Pharmingen Cat. No. 559943, 1:100 dilution), CD49d (BioLegend Cat. No. 304304, 1:20 dilution), and Band3 (Gift from X. An, 1:100 diltuion) (Supplementary Table 1). To quantify erythroblast enucleation, 1.5–5 × 10⁵ CD34+-derived erythroid cells were incubated with Hoechst 33342 (Millipore Sigma Cat. No. B2261, 1:1000 dilution) for 20 min at 37 °C, fixed with 0.05% glutaraldehyde, and permeabilized with 0.1% Triton X-100.

Newby G.A., Yen J.S., Woodard K.J., Mayuranathan T., Lazzarotto C.R., Li Y., Sheppard-Tillman H., Porter S.N., Yao Y., Mayberry K., Everette K.A., Jang Y., Podracky C.J., Thaman E., Lechauve C., Sharma A., Henderson J.M., Richter M.F., Zhao K.T., Miller S.M., Wang T., Koblan L.W., McCaffrey A.P., Tisdale J.F., Kalfa T.A., Pruett-Miller S.M., Tsai S.Q., Weiss M.J, & Liu D.R. (2021). Base editing of hematopoietic stem cells rescues sickle cell disease in mice. Nature, 595(7866), 295-302.

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Flow Cytometry Immune Cell Profiling

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Whole blood samples were obtained in EDTA tubes. Blood was divided into aliquots and treated with Fc Block, Live/Dead (Invitrogen) and the following antibodies (5 μL per sample): CD14, CD16, CD86, CD54, CD163, CD18, CD36, and CD49d (BioLegend). Samples were incubated in the dark, on ice, for 30 minutes with gentle agitation. The tubes were then treated with 1‐step Fix/Lyse Solution (eBioscience) and incubated for an additional 15 minutes at room temperature. Samples were then centrifuged at 500g for 5 minutes, washed with flow buffer, centrifuged again at 500g for 5 minutes, resuspended, and analyzed by flow cytometry (250 000 events per sample, BD FACSVerse, Becton, Dickinson and Company). Analysis was conducted using FlowJo software (FlowJo, LLC). Quantitative data are described by mean fluorescence intensity (MFI) for each marker.

Schaller M.S., Chen M., Colas R.A., Sorrentino T.A., Lazar A.A., Grenon S.M., Dalli J, & Conte M.S. (2020). Treatment With a Marine Oil Supplement Alters Lipid Mediators and Leukocyte Phenotype in Healthy Patients and Those With Peripheral Artery Disease. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 9(15), e016113.

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Multiparametric Flow Cytometry Analysis

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Singe cell preparations were isolated from indicated tissue sites and flow stained. Fluorochrome-conjugated mAbs specific to mouse CD4 (clone GK1.5), CD19 (clone 6D5), CD25 (clone PC61.5), CD29 (clone HMβ1-1), CD39 (clone 24DMS1), CD40L (clone MR1), CD44 (clone IM7), CD49d (clone R1-2), CD62L (clone MEL-14), CD69 (clone H1.2F3), CD73 (Clone eBioTY/11.8), CD103 (clone 2E7), CD127 (clone A7R34), 4-1BB (clone 17B5), GITR (clone DTA-1), ICOS (clone 7E.17G9), LAG-3 (clone eBioC9B7W), LAP (clone TW7-16B4), PD-1 (clone 29F.1A12) and Thy1.1 (clone OX-7) (1:400 dilution, Biolegend or eBioscience) were used in various combinations for cell surface staining. Biotin-labeled anti-VISTA mAb was provided by Dr. Li Wang (Medical College of Wisconsin). The Fc receptor was blocked by CD16/32 antibody (Biolegend). For Foxp3 (1:400 dilution; clone FJK-16s, eBioscience) and CTLA-4 (1:400 dilution; clone UC10-4B9, Biolegend), cells were treated by eBioscience intracellular fixation/permeabilization buffer set and intracellular staining was performed. Stained cells were assayed by MACSQuant Analyzers (Miltenyi, Bergisch Gladbach, Germany).

Wang Y., Telesford K.M., Ochoa-Repáraz J., Haque-Begum S., Christy M., Kasper E.J., Wang L., Wu Y., Robson S.C., Kasper D.L, & Kasper L.H. (2014). An intestinal commensal symbiosis factor controls neuroinflammation via TLR2-mediated CD39 signaling. Nature communications, 5, 4432.

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Multiparametric Flow Cytometry Analysis

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Immunoassay Antibodies for Protein Analysis

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The following antibodies were used for immunoassays: p‐S6 Ser240/244 (Cell Signalling, 5364 and 6520), S6 (Cell Signalling, 2217), p‐4EBP1 Thr37/46 (Cell Signalling, 2855), 4EBP1 (Cell Signalling, 9644), p‐AKT Ser473 and Thr308 (Cell Signalling, 9271 and 9275), AKT (Cell Signalling, 4691), p‐STAT3 Tyr705 (Cell Signalling, 4113 and BioLegend, 651021), STAT3 (Cell Signalling, 9139), p‐P65 (Cell Signalling, 3031 and 5733), P65 (Cell Signalling, 8242), Vinculin (Sigma, V4505), Iba1 (Wako, 019‐19741), CD11b (eBiosciences, 17‐0112‐82) and (Abcam, ab8878), CD45 (BioLegend, 103105), Ki67 (BioLegend, 652413), Granzyme b (BioLegend, 337221) and Perforin (BioLegend, 154406). IFNʏ (BioLegend, 505826), CD49d (BioLegend, 103618 and 304313), CD44 (BioLegend, 10349), CD62L (BioLegend, 104438), CD3 (BioLegend, 152303), CD4 (BioLegend, 100427), CD8 (BioLegend, 100722 and 100732), PU.1 (Cell Signalling, 2258), c‐kit (Abcam, ab212518), CD41 (BioLegend, 303702), CD235a (Life Technology, 14‐9987‐82), P2RY12 (Atlas, HPA014518 and 848006), TREM2 (Abcam, ab86491),TMEM119 (Sigma, HPA051870), MHCII (BioLegend, 107607), F480 (BioLegend, 123130), Sox2 (Santa cruz, sc‐17320) and NANOG (BioLegend 16H3A38).

Dumas A.A., Pomella N., Rosser G., Guglielmi L., Vinel C., Millner T.O., Rees J., Aley N., Sheer D., Wei J., Marisetty A., Heimberger A.B., Bowman R.L., Brandner S., Joyce J.A, & Marino S. (2020). Microglia promote glioblastoma via mTOR‐mediated immunosuppression of the tumour microenvironment. The EMBO Journal, 39(15), e103790.

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CITE-Seq Analysis of PBMC

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The following human Total-seq Biolegend antibodies were used for the CITE-Seq of PBMC: CD19 (cat: 302259), CD5 (cat: 300635), CD3 (cat: 300475), CD4 (cat: 300563), CD56 (cat: 392421), CD8a (cat: 344751), CD14 (cat: 301855), CD69 (cat: 310947), CD279(PD-1) (cat:329955), CD49d (cat: 96538), CD20 (cat: 302359), control isotype IgG1(cat: 400199).

Cadot S., Valle C., Tosolini M., Pont F., Largeaud L., Laurent C., Fournie J.J., Ysebaert L, & Quillet-Mary A. (2020). Longitudinal CITE-Seq profiling of chronic lymphocytic leukemia during ibrutinib treatment: evolution of leukemic and immune cells at relapse. Biomarker Research, 8, 72.

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Comprehensive Antibody Panel Profiling

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Antibodies were selected based on expected differences in expression levels among the 5 cell lines. The antibodies that were used belong to three different categories providing information of tissue origin, recognizing antigens involved in cell adhesion and potential drug targets. Anti-human EpCAM/CD326, Her2 and EGFR antibody were provided by Immunicon Corp. (Huntingdon Valley, Philadelphia, USA). Anti-human CD3, CD8a, CD11c, CD14, CD19, CD20, CD25, CD33, CD45, CD56, CD61, CD66b, CD105, CD123, CD140a, CD146, CD235a, CD71, CD117, CD221, CD227, CD261, CD262, CD309, Her3, CD24, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, CD104, CD106, CD113, CD144, CD166, CD324, CD334 antibodies were purchased from Biolegend Inc. (San Diego, CA, USA). Anti-human CD113 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-human CD103 was purchased from BD Biosciences (San Jose, CA, USA). Anti-human serum albumin (HSA) antibody was used as a negative control in the SPRi experiments and was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Anti-mouse IgG PE was purchased from Abcam (Cambridge, UK) and was used for staining of unlabeled antibodies in flow cytometry and as a negative control in the SPRi experiments.

Stojanović I., Ruivo C.F., van der Velden T.J., Schasfoort R.B, & Terstappen L.W. (2019). Multiplex Label Free Characterization of Cancer Cell Lines Using Surface Plasmon Resonance Imaging. Biosensors, 9(2), 70.

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Multiparametric Flow Cytometry for Cell Identification

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Cells were analyzed and sorted by flow cytometers FACS Aria 2 and Calibur (BD Biosciences), and the data were analyzed with FlowJo software (Tree Star). Cells were stained by the following antibodies: B220 (eBioscience, RA3-6B2), CD3 (eBioscience, 145-2C11), CD4 (eBioscience, GK1.5), CD8 (eBioscience, 53-6.7), CD19 (eBioscience, eBio1D3), CD31 (BD or BioLegend, MEC13.3), CD34 (eBioscience, RAM34), CD41 (BD or eBioscience, MWReg30), CD43 (BD, S7), CD44 (eBioscience or BioLegend, IM7), CD45 (eBioscience, 30-F11), CD45.1 (eBioscience, A20), CD45.2 (eBioscience, 104), CD47 (eBioscience, miap301), CD49d (Biolegend, R1-2), CD93 (eBioscience, AA4.1), CD133 (Biolegend, 315-2C11), CD146 (BD, ME-9F1), CD201 (eBioscience, eBio1560), CD304 (Biolegend, 3E12), Flk1 (eBioscience, Avas12a1), Flk2 (Biolegend, A2F10), F4/80 (Biolegend, 93), Kit (eBioscience, 2B8), Ly-6G (BioLegend, 1A8), Mac-1 (eBioscience, M1/70), Sca-1 (eBioscience, D7), Ter119 (BD, TER-119), and 7-amino- actinomycin D (7-AAD; eBioscience).

Li Y.Q., Gong Y., Hou S., Huang T., Wang H., Liu D., Ni Y., Wang C., Wang J., Hou J., Yang R., Yan J., Zhang G., Liu B, & Lan Y. (2021). Spatiotemporal and Functional Heterogeneity of Hematopoietic Stem Cell-Competent Hemogenic Endothelial Cells in Mouse Embryos. Frontiers in Cell and Developmental Biology, 9, 699263.

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Comprehensive Immunophenotyping by Flow Cytometry

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The following antibodies were used in an appropriate combination of fluorochromes: B220 (clone RA3–6B2, BioLegend), Bcl-6 (clone K112–91, BD Biosciences), CD4 (clone GK1.5, BioLegend), CD8α (clone 53–6.7, BioLegend), CD11a (clone M17/4, BioLegend), CD19 (clone 1D3/CD19, BioLegend), CD21 (Clone 7E9, BioLegend), CD23 (Clone B3B4, BioLegend), CD49d (clone R1–2, BioLegend), CD86 (Clone GL-1, BioLegend), CXCR5 (clone L138D7, BioLegend), FoxP3 (clone FJK-16s, eBioscience), GL7 (clone GL7, BioLegend), IFN-γ (clone XMG1.2, BioLegend), IgD (clone 11–26c.2a, BioLegend), IL-2 (clone JES6–5H4, BioLegend), IL-5 (clone TRFK5 BioLegend), IL-17A (clone TC11–18H10.1, BioLegend), PD-1 (clone 29F.1A12, BioLegend), T-bet (clone 4B10, BioLegend), TNF-α (clone MP6-XT22, BioLegend), and appropriate isotype controls.

Pardy R.D., Gentile M.E., Carter A.M., Condotta S.A., King I.L, & Richer M.J. (2022). An Epidemic Zika Virus Isolate Drives Enhanced T Follicular Helper Cell and B Cell-Mediated Immunity. Journal of immunology (Baltimore, Md. : 1950), 208(7), 1719-1728.

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