Anti cd38 pe

Manufactured by BioLegend

Sourced in United States

Anti-CD38-PE is a fluorescently-labeled antibody that binds to the CD38 cell surface protein. It can be used for the identification and enumeration of CD38-expressing cells in flow cytometry applications.

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13 protocols using anti cd38 pe

Multiparametric Flow Cytometry for Cell Purity

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Both primary cells and cell lines were tested using multiparametric flow cytometry for their purity.
Anti-CD10-PerCP/Cy5.5 (Cat no. 312215), anti-CD19-APC (Cat no.392503), anti-CD20-FITC (Cat no. 302303), and anti-CD38-PE (Cat no 356603) were purchased from Biolegend. Fluorescent viability dye, eFlour780, was purchased from ThermoFisher Scientific (Cat no. 65–0865-14). Cells were stained with individual antibodies (quantity as recommended by the manufacturer) in cold PBS containing 0.5% fetal calf serum and the fluorescent viability dye (1:1000 dilution) for 20 minutes. Cells were maintained at a density of one million cells per 100 ml of buffer. Cells were then washed three times with 200 μl of the buffer and resuspended at the above density. Control (unstained) cells were stained with just the fluorescent viability dye. Flow cytometry was performed to analyze the stained and control cells on a MACSQuant Analyzer, and the acquired data were further analyzed and plotted using FlowJo. At least 30,000 cells were acquired from each sample.

Paidi S.K., Raj P., Bordett R., Zhang C., Karandikar S.H., Pandey R, & Barman I. (2021). Raman and quantitative phase imaging allow morpho-molecular recognition of malignancy and stages of B-cell acute lymphoblastic leukemia. Biosensors & bioelectronics, 190, 113403.

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Detailed PBMC Phenotyping Protocol

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Peripheral blood mononuclear cells (PBMCs), separated by density gradient centrifugation with Lymphocyte Separation Medium (Life Technology), were resuspended with RPMI medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 1 × 10⁶ cells/mL, and then incubated with various combinations of monoclonal antibodies and isotype controls. The following antibodies were used to identify different B cell subsets: anti-CD19-PerCP, anti-CD27-FITC, anti-IgD-BV510, anti-CD38-PE (all from Biolegend, San Diego, CA, USA), and anti-IgM-APC (BD Bioscience, San Diego, CA, USA) (Supplementary Figure S1 and Table S1).
The cells were acquired using FACSCelesta (Beckton-Dickinson, San Jose, CA, USA) and analyzed using FlowJo software. Isotype control antibodies and single-stained samples were used to periodically check the settings and gates on the flow cytometer. After the acquisition of 100,000 to 200,000 events, lymphocytes were gated based on forward and side scatter properties after the exclusion of dead cells and doublets.

Kasahara T.D, & Gupta S. (2024). IgD+IgM− B Cells in Common Variable Immunodeficiency. Pathogens, 13(2), 136.

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Multiparametric Flow Cytometry Analysis of MM Cells

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Following drug treatment, MM cells were pelleted and resuspended in flow cytometry buffer (1× PBS with 1% FBS) then incubated with human FcR blocking reagent (Miltenyi Biotec, # 130‐059‐901) before staining with diverse fluorescent dye‐conjugated monoclonal antibodies: anti‐CD126 (IL‐6Rα)/APC (#352806 Biolegend, dilution 1/50), anti‐CD38/BV421 (#303526 Biolegend, diluted 1/200), anti‐CD38/PE (#356604 Biolegend, diluted 1/200), anti‐CD138/APC (#130‐117‐395 Miltenyi Biotec, diluted 1/100), anti‐CD138/APC‐AF750 (#352316 Biolegend, diluted 1/100), anti‐CD40/FITC (#334306 Biolegend, diluted 1/25), and anti‐BCMA/PerCp‐Cy5.5 (#357510 Biolegend, diluted 1/25). Cells were stained for 15 min at 4°C, then washed and filtered before data acquisition by Cytoflex flow cytometer (Beckman Coulter). Data were analyzed with the FlowJo software (Tree Star, Ashland, OR). Mean fluorescence intensities (MFI) of isotype controls recommended by the Ab supplier were systematically subtracted to that of each stained sample. Cell viability was assessed by Annexin V exposure and PI incorporation using the FITC‐Annexin V/PI kit from Miltenyi Biotec (#130‐092‐052) following the manufacturer’s protocol. Live cells were characterized as Annexin V^‐ PI^‐, apoptotic cells as Annexin V⁺ PI⁻, and dead cells as Annexin V⁺ PI⁺ and Annexin V⁻ PI⁺ as shown in Appendix Fig S1.

Domenger A., Choisy C., Baron L., Mayau V., Perthame E., Deriano L., Arnulf B., Bories J., Dadaglio G, & Demangel C. (2022). The Sec61 translocon is a therapeutic vulnerability in multiple myeloma. EMBO Molecular Medicine, 14(3), e14740.

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Activation and Analysis of NK Cells

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An isolation kit (Stemcell, CAN) was used to isolate the NK cells, which were activated by Phorbol 12-myristate 13-acetate (PMA)/ionomycin (BioLegend, USA) and protein transport inhibitor GolgiStop (BD Biosciences) in 96-well U-bottomed plates with 5% CO2 at 37 °C for 6 h. First, the NK cells were collected, and Fixable viability stain 620 (BD Pharmingen, USA) was added to exclude dead cells. Antibodies for cell surface staining included anti-CD3-APC-CY7, anti-CD56-PE-Cy7, anti-CD38-PE, anti-CD39-FITC (BioLegend, USA) and were incubated for 20 mins. Next, the cells were fixed and washed by Fixation/Permeabilization solution (BD Biosciences, USA) and intracellularly labeled by anti-IL-10-APC and anti-TGF-β-BV421 (BD Biosciences, USA) at 4 °C for 20 mins. Finally, the cells were measured by LSR II Fortessa cytometer (BD Biosciences, USA).

Qian S., Xiong C., Wang M., Zhang Z., Fu Y., Hu Q., Ding H., Han X., Shang H, & Jiang Y. (2022). CD38+CD39+ NK cells associate with HIV disease progression and negatively regulate T cell proliferation. Frontiers in Immunology, 13, 946871.

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Cytotoxicity Assessment of Compounds in Tonsillar Tissues

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To assess the cytotoxicity of (1), (2), and (5) in human tonsillar tissues after 12 days of culture, cells isolated from untreated tissue blocks and from those treated with compounds were stained with combinations of the following fluorescence- labeled monoclonal antibodies: anti– CD3-QD605, anti–CD4-QD655, anti–CD8-QD705, anti–CD25-APC, anti–CD38-PE, anti–HLA-DR-APC-Cy7, anti–CXCR4-Brilliant violet 421, anti–CCR5-PR-Cy5 anti–CD45RA-FITC, and anti–CCR7-PE-Cy7 (Caltag Laboratories; Biolegend). Detection and enumeration of HIV-1– infected cells were performed with intracellular staining by means of anti–p24-PE (Beckman Coulter). Data were acquired and analyzed as described elsewhere (Grivel and Margolis, 2009 ). We quantified cell depletion using Trucount beads (Becton Dickinson) for volumetric control and normalized cell numbers by tissue-block weights.

Vanpouille C., Khandazhinskaya A., Karpenko I., Zicari S., Barreto-de-Souza V., Frolova S., Margolis L, & Kochetkov S. (2014). A NEW ANTIVIRAL: CHIMERIC 3TC-AZT PHOSPHONATE EFFICIENTLY INHIBITS HIV-1 IN HUMAN TISSUES EX VIVO. Antiviral research, 109, 125-131.

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SIV-Infected Rhesus Macaque PBMC Immunophenotyping

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For the memory and differentiation panel, similar steps were carried out during surface staining. This involved the addition of anti-CD10 APC Cy7 Clone H110a (BioLegend), anti-CD3 AF700 Clone SP34-2 (BD Biosciences), anti-CD4 BV605 Clone L200 (BD optibuild, BD Biosciences), anti-CD45RA PE-CF594 Clone 5H9 (BD Horizon, BD Biosciences), anti-human CCR7 PE Cy7 Clone 3D12 (BD Biosciences) monoclonal antibodies. Lastly, to determine the distribution of IA markers within the studied CD4⁺ T cell subsets, anti- CD38 PE (NHP reagent Resource) and anti-PD-1 PECy5.5 Clone EH12.2H7 9 (BioLegend) were added to the PE and PE Cy5.5 channels of the memory and differential panel and similar procedures for flow cytometry surface staining followed. In a separate panel, anti-CD28 APC Clone CD28.2 (BD Biosciences) and anti-CD95 PerCP-Cy^TM 5.5 Clone DX2 (BD Pharmingen^TM, BD Biosciences) monoclonal antibodies were included. This panel was used to characterize memory and differentiation status in PBMCs obtained from SIV infected RMs treated with RA of blood. Following this, cells were fixed with 1% PFA.

Olwenyi O.A., Acharya A., Routhu N.K., Pierzchalski K., Jones J.W., Kane M.A., Sidell N., Mohan M, & Byrareddy S.N. (2020). Retinoic Acid Improves the Recovery of Replication-Competent Virus from Latent SIV Infected Cells. Cells, 9(9), 2076.

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Multiparametric Flow Cytometry Analysis of Immune Cell Subsets

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The PBMCs or TILs were stained with the following fluorochrome-labeled monoclonal antibodies (mAbs) at 4°C for 30 min in the dark: Anti-CD3-PerCP (1:100; cat. no. 300428; BioLegend, Inc.), anti-CD8-FITC (1:100; cat. no. 11-0086.42; eBioscience; Thermo Fisher Scientific, Inc.), anti-PD-1-PE/Cy7 (1:100; cat. no. 329918; BioLegend, Inc.), anti-CD38-PE (1:100; cat. no. 4322550; Invitrogen; Thermo Fisher Scientific, Inc.) and anti-CD101-APC (1:100; cat. no. 331007; BioLegend, Inc.). Isotype control antibodies were used to ensure accurate compensation and to set gates. The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo 10 software (FlowJo LLC).

Zhou J., Wang W., Liang Z., Ni B., He W, & Wang D. (2020). Clinical significance of CD38 and CD101 expression in PD-1+CD8+ T cells in patients with epithelial ovarian cancer. Oncology Letters, 20(1), 724-732.

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Tonsil B Cell Immunophenotyping

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Tonsil mononuclear cells were thawed, washed, and counted as described above. To stain apoptotic and necrotic cells, cells were incubated with Zombie NIR fixable viability dye (Biolegend) for 15 min at room temperature. Cells were washed, resuspended in a surface stain cocktail containing anti-IgD-FITC, anti-CD38-PE, anti-CD27-PE/Cy7, anti-CD19-APC, anti-CD3-APC/Cy7, and anti-CD14-APC/Cy7 (all from Biolegend), and incubated for 45 min on ice. Subsequently, cells were washed two times, passed through a 35 µm nylon mesh and sorted on a BD FACS Aria II at the Harvard Microbiology and Immunobiology (MBIB) Flow Cytometry Core. Lymphocytes were electronically gated according to their forward scatter and side scatter properties, negatively gated to exclude viability dye⁺, CD3⁺, and CD14⁺ cells, and positively gated for B cells (CD19⁺). B cell subsets were subsequently gated as follows: IgD ⁺CD27^- cells (Naive); IgD^- CD38^hi CD27^+/- (GC); IgD^- CD38^lo/mid CD27 ⁺ (Memory) (See also Fig. 1b and Supplementary Fig. 1). The CD27 gate was set based on a PE/Cy7 FMO gating control. Sorted cells were pelleted, washed 2× with PBS, then lysed in Buffer RLT (Qiagen, for RNA analysis), 2% NP-40 lysis buffer (for western blot), or snap frozen in an isopropanol/dry ice slurry (for glycomic analysis).

Giovannone N., Liang J., Antonopoulos A., Geddes Sweeney J., King S.L., Pochebit S.M., Bhattacharyya N., Lee G.S., Dell A., Widlund H.R., Haslam S.M, & Dimitroff C.J. (2018). Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans. Nature Communications, 9, 3287.

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Murine and Human Monocyte Phenotyping

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Murine monocytes and human monocytes were harvest at day 5 or day 1 and blocked FcBlock (anti-mouse CD16/32, BD Biosciences or Human TruStain FcX, Biolegend) followed by stanning with mouse anti-CD11b (APC-Cy7, Biolegend), anti-Ly6C (PE-Cy7, Biolegend), anti-PD-L1 (APC, Biolegend), anti-CD38 (PE, Biolegend), anti-CD86 (FITC, Biolegend) or human anti-CD66b (FITC, Biolegend), anti-CD14(PE-Cy7, Biolegend), anti-CD16 (APC-Cy7, Biolegend), anti-CD86 (APC, Biolegend), anti-PD-L1 (PE, Biolegend), anti-CD38 (APC, Biolegend) for 30 minutes. For monocyte-T cell coculture, T cells were blocked and stained with mouse anti-CD4 (FITC, Biolegend), anti-CD8 (APC, Biolegend), and anti-CD69 (APC-Cy7, Biolegend). Stained cells were washed with FACS buffer and resuspended into the FACS buffer containing Propidium Iodide (Invitrogen). MitoTracker™ Deep Red FM kit (Invitrogen) was used to detect mitochondrial potential as described by the manufacturer's instructions. Antibodies labeled samples were analyzed with a FACSCanto II (BD Biosciences), and data were analyzed with FlowJo (BD Life Sciences).

Wu Y., Caldwell B., Wang J., Zhang Y, & Li L. (2024). Alleviation of monocyte exhaustion by BCG derivative mycolic acid. iScience, 27(2), 108978.

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Isolation and Characterization of Live NR1+ B Cells

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Fresh CSF was transported at 4°C and centrifuged at 1,200 rpm for 10 min immediately. The cells were suspended with 500 μl cryopreservation solution (90%FBS +10%DMSO) and stored at −80°C. Recombinant Protein NR1 (OriGene) was labeled by lightning-link ®FITC (Expedeon) through incubation for 3 h in dark conditions. Before flow cytometry, the cells were thawed into 1 ml FBS (4°C) +15 ml 1640 medium (4°C) and centrifuged at 250 g for 10 min. The supernatant was discarded. Then 1 ml Cell Staining Buffer (Biolegend) and fluorescent antibodies were added. All kinds of antibodies (anti-CD20-percp/Cy5.5, anti-CD27-APC, anti-CD38-PE, Biolegend; NR1-FITC) were added with 1 μl to the cell suspension.
4',6-diamidino-2-phenylindole (DAPI) (3 μmol/L, add 500 μl/tube) staining for about 5 min. Cells were sorted into 8-strip PCR tubes, and each tube contained 4 μl ice-cold cell lysis buffer: 1.86 μl nuclease-free water, 1 μl Oligo dT18 (10 μmol/L), 0.1 μl RNase inhibitor (4U, Applied Biosystems), 0.04 μl Triton X-100 (100 ml/L, Sigma), and 1 μl dNTPmix (10 mm). Using BD FACSAria IIIu flow cytometry, target cells (CD20+NR1+) with negative and weak positive DAPI (indicating the cells were still alive) were selected and placed in liquid nitrogen immediately.

Feng J., Fan S., Sun Y., Zhang Z., Ren H., Li W., Cui L., Peng B., Ren X., Zhang W., Guan H, & Wang J. (2020). Study of B Cell Repertoire in Patients With Anti-N-Methyl-D-Aspartate Receptor Encephalitis. Frontiers in Immunology, 11, 1539.

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