Peripheral B cells were purified from the blood of patients and control donors by positive selection using CD20 magnetic beads (Miltenyi Biotec). For sorting, enriched B cells were stained with FITC-anti-IgM, PE-anti-IgG, PE-Cy7-anti-CD10, APC-anti-CD21, Pacific Blue-anti-CD19, and PercP-Cy5.5-anti-CD27 (all from BioLegend) (gating strategy is shown in fig. S21 ). Single- or batch-sorted CD19+CD21loCD10+IgMhiCD27− new emigrant and CD19+CD21+CD10−IgM+CD27− mature naïve B cells from patients, heterozygous relatives, and HDs were sorted on a FACSAria flow cytometer (Becton Dickinson, Mountain View, CA) into 96-well polymerase chain reaction (PCR) plates or 5-ml round-bottom polystyrene test tubes, respectively. For phenotyping, enriched B cells were stained with FITC-anti-IgM, PE-anti-CD69, PE-Cy7-anti-CD10, APC-anti-CD86, PercP-Cy5.5-anti-CD27, APC-Cy7-anti-CD19 (all from BioLegend), and V450-anti-CD21 (BD Bioscience) (gating strategy is shown in fig. S22 ).
Anti igm fitc
Manufactured by BioLegend
Sourced in United States
Anti-IgM FITC is a fluorescently-labeled antibody that specifically binds to the mu heavy chain of immunoglobulin M (IgM). This product is designed for use in flow cytometry and other immunoassays to detect and quantify IgM-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd21 apc, by BioLegend (2 mentions)
Anti cd19 apc cy7, by BioLegend (2 mentions)
Anti pd 1 pe, by BioLegend (2 mentions)
Anti cd25 pe, by BioLegend (2 mentions)
Anti cd8 apc, by BioLegend (2 mentions)
Anti cd86 apc, by BioLegend (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Cd20 magnetic beads, by Miltenyi Biotec (1 mentions)
Anti cd19 pacific blue, by BioLegend (1 mentions)
Pe cy7 anti cd10, by BioLegend (1 mentions)
Percp cy5.5 anti cd27, by BioLegend (1 mentions)
Anti cxcr5 apc, by BioLegend (1 mentions)
Anti cd62l fitc, by BioLegend (1 mentions)
Fitc anti icos, by BioLegend (1 mentions)
Apc anti tim 3, by BioLegend (1 mentions)
Fitc anti annexin 5, by BioLegend (1 mentions)
Anti cd4 fitc, by BioLegend (1 mentions)
Anti igd pe, by BioLegend (1 mentions)
Facscanto 10c cytometer, by BD (1 mentions)
Trucount tube, by BD (1 mentions)
8 protocols using anti igm fitc
1
Sorting and Phenotyping of B Cell Subsets
2
Multiparametric Flow Cytometry Panel
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For flow cytometry, we used FITC anti-CD4, APC anti-CD8, FITC anti-IgM, APC anti-CXCR5, PE anti-PD1, FITC anti-CD62L, PE anti-CD25, APC anti-LAG-3, APC anti-TIM-3, PE anti-PD-1 and FITC anti-ICOS, FITC anti-Annexin V and APC anti-FoxP3 antibodies (all from BioLegend).
3
Comprehensive Immune Profiling by Flow Cytometry
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All flow cytometric analyses were performed using the FACSCanto 10c cytometer (BD Biosciences, Heidelberg, Germany) and BD FACSDiva Software version 8.0.1. Cellular immune status (Panel 1), T cell activation and exhaustion (Panel 2) and B cell phenotype (Panel 3) were determined using specific markers for innate leukocytes, T cells and B cells. Briefly, cell frequencies and total cell numbers in whole blood samples were analyzed on a single-cell platform using TruCount™ tubes (BD Biosciences). Cells were stained for 30 min at room temperature using anti-CD45 allophycocyanin-H7 (APC-H7) or AlexaFluor 700 (AF-700), anti-CD3 fluorescein isothiocyanate (FITC), anti-CD8 allophycocyanin (APC), anti-CD4 peridinin chlorophyll protein complex (PerCP), anti-CD19 AF-700 or Brilliant Violet (BV510), anti-CD56 phycoerythrin (PE), anti-CD14 Brilliant Violet (BV510), anti-CD45RA BV605 and anti-CD62L BV421 or BV510, anti-CD20 APC-cyanine 7 (APC-Cy7), anti-CD27 BV421, anti-CD38 APC, anti-CD24 PerCP, anti-IgD PE, anti-IgM FITC and anti-PD-1 PE monoclonal antibodies (BioLegend and BD Biosciences) either before (Panels 1 and 2) or after (Panel 3) lysis of erythrocytes using 1x Lysing Solution according to the manufacturer’s instructions (Panels 1 and 2: BD Biosciences, Panel 3: Beckman Coulter, Brea, CA, USA).
4
Comprehensive Multiparametric Flow Cytometry
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Cell surface markers were stained with these specific antibodies: anti-CD19-PE, anti-CD3-APC, anti-CD4-PE, anti-CD8-FITC, 7-AAD, and anti-annexin-V-FITC (eBioscience); anti-CD19-APC, anti-B220-Per-cy5.5, anti-IgM-FITC, anti-AA4.1-PE, anti-CD23-eFluor647, and anti-CD8-APC (Biolegend).
For ex vivo analysis, cells were stimulated with 25 ng/mL PMA (Sigma-Aldrich) and 1 g/mL ionomycin (Sigma-Aldrich) in the presence of 0.66 μL/mL Golgistop (BD PharMingen) for 6 h at 37 °C, 5 % CO2. Intracellular staining of IFN-γ and IL-4 was performed using Transcription Factor Staining Buffer Set (eBioscience). Data was collected by FACS Calibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR).
For cell quantization, blood sample (100 μL) was stained with the specific antibodies: anti-CD19-FITC, anti-CD3-APC, anti-CD4-FITC, anti-CD8-FITC, anti-CD11c-APC, anti-F4⁄80-FITC, and anti-CD11b-APC. Erythrocytes were then lysed with Cal-lyse Lysing Solution (Invitrogen). After thoroughly mixing with 100 μL of Caltag Counting Beads (Invitrogen), 10,000 beads were acquired in the FACS Calibur flow cytometer for each sample.
For ex vivo analysis, cells were stimulated with 25 ng/mL PMA (Sigma-Aldrich) and 1 g/mL ionomycin (Sigma-Aldrich) in the presence of 0.66 μL/mL Golgistop (BD PharMingen) for 6 h at 37 °C, 5 % CO2. Intracellular staining of IFN-γ and IL-4 was performed using Transcription Factor Staining Buffer Set (eBioscience). Data was collected by FACS Calibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR).
For cell quantization, blood sample (100 μL) was stained with the specific antibodies: anti-CD19-FITC, anti-CD3-APC, anti-CD4-FITC, anti-CD8-FITC, anti-CD11c-APC, anti-F4⁄80-FITC, and anti-CD11b-APC. Erythrocytes were then lysed with Cal-lyse Lysing Solution (Invitrogen). After thoroughly mixing with 100 μL of Caltag Counting Beads (Invitrogen), 10,000 beads were acquired in the FACS Calibur flow cytometer for each sample.
5
Isolation and Characterization of SARS-CoV-2-Specific Memory B Cells
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B cells were isolated from PBMCs of convalescent individuals with COVID-19 using immunomagnetic negative selection (EasySep Human B Cell Enrichment Kit, STEMCELL, 17954). In this process, Tetrameric Antibody Complexes and dextran-coated magnetic particles targeted and removed non-B cells. Following isolation, B cells underwent staining with anti-CD19-APC (BioLegend, 302212), anti-IgD-FITC (BioLegend, 348206), anti-IgM–FITC (BioLegend, 314506) phenotyping antibodies, and biotinylated SARS-CoV-2 (BA.4/5) Spike RBD antigen. Viability was assessed using 7-AAD Stain (Invitrogen, 00699342). Class-switched memory B cell-antigen complexes (CD19+Ag+IgM-IgD-7-AAD-) were then detected with a PE-labeled streptavidin conjugate (BioLegend,127807), and target memory B cells were isolated via flow-cytometric sorting using a BD FACSAira Fusion (BD Biosciences). Subsequently, cells were cultured and activated in human B cell expansion medium for 8 days (ImmunoCultTM Human B Cell Expansion Kit, STEMCELL, 1000645). Flow-cytometric data analysis was conducted using FlowJo version 10.8.1 (Tree Star).
6
Multiparametric Flow Cytometry Panel
7
Comprehensive Multiparameter Flow Cytometry
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Supernatants were removed and cell pellets were resuspended in 200 μL of staining buffer containing 1 μL each of anti-CD38-APC (BioLegend, clone HIT2), anti-CD13-PE-Cy7 (BioLegend, clone WM15), anti-CD21-PE-Dazzle (BioLegend, clone Bu32), anti-CD19-APC-efluor-780 (Invitrogen, clone HIB19), anti-IgD-PerCP-Cy5.5 (Biolegend, clone IA6–2), anti-IgM-FITC (Biolegend, clone MHM-88), anti-CD27-BV510 (Biolegend, clone M-T271), anti-CD11c-Alexa700 (BioLegend, clone Bu15). Staining buffer also contained Delta-S1-PE and S1-BV421 tetramers.
8
Indirect Immunofluorescence for ANA-IgM
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Kallestad HEp-2 slides (BIO-RAD, #26101) were used to asses reactivity of purified homemade IgM or pulldown serum IgM to nuclear antigens (ANA). Approximately 10 µg per sample were applied onto the HEp-2 slides. Anti-IgM-FITC (Biolegend, #314506) was used for detection of ANA-IgM. Stained HEp-2 slides were analyzed using fluorescence microscope DMi8 (Leica) and Leica Application Suite X (LAS X) software (Leica).
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