LN biopsy samples were minced and evaluated by two-color flow cytometry after staining in phosphate-buffered-saline without calcium chloride magnesium chloride (PBS (−)) with the following monoclonal antibodies: anti-CD19-phycoerythrin (PE) (BioLegend, San Diego, CA, USA), anti-CD27-PE (BioLegend), anti-CD3-PE (BioLegend), anti-CD20-fluorescein isothiocyanate (FITC) (BioLegend), and anti-CD38-FITC (BioLegend). BiPSCs were also evaluated by two-color flow cytometry before and after the induction of hematopoietic differentiation using the following monoclonal antibodies: anti-CD19-PE, anti-CD27-PE, anti-CD34-PE (BioLegend), anti-CD20-FITC, anti-CD38-FITC, anti-CD43-FITC (BioLegend), and anti-CD45-FITC (BioLegend). Immunofluorescence of the labeled cell membrane was evaluated using a BD FACSCANTO II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Anti cd27 pe
Manufactured by BioLegend
Sourced in United States
Anti-CD27-PE is a fluorescently labeled antibody that binds to the CD27 cell surface receptor. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on various immune cell types. This antibody can be used for the identification and analysis of CD27-positive cells in flow cytometry applications.
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Lab products found in correlation
Anti cd3 fitc, by BioLegend (3 mentions)
Anti cd19 pe, by BioLegend (2 mentions)
Anti cd8 fitc, by BioLegend (2 mentions)
Anti cd56 pe, by BioLegend (2 mentions)
Anti cd11b pe, by BioLegend (2 mentions)
Mouse recombinant il 21, by Thermo Fisher Scientific (2 mentions)
Anti nkp46 pe cy7, by BioLegend (2 mentions)
Isotype matched control antibody, by Thermo Fisher Scientific (2 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
Allophycocyanin klrg1, by BioLegend (2 mentions)
Facscanto 2, by BD (1 mentions)
Anti cd45 fitc, by BioLegend (1 mentions)
Anti cd3 pe, by BioLegend (1 mentions)
Anti cd43 fitc, by BioLegend (1 mentions)
Anti cd38 fitc, by BioLegend (1 mentions)
Anti cd20 fitc, by BioLegend (1 mentions)
Anti cd34 pe, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Anti cd19 pacific blue, by BioLegend (1 mentions)
Anti cd10 apc, by BioLegend (1 mentions)
8 protocols using anti cd27 pe
1
Characterization of B-cell Differentiation
2
Multiparametric Flow Cytometric Analysis of B Cell Subsets
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Surface staining of PBMCs was performed with the following flourochrome conjugated monoclonal antibodies: anti-CD19 Pacific Blue, anti-CD10 APC, anti-CD20 APC-Cy7, anti-CD21 FITC, anti-CD27 PE (all Biolegend). Viability Live/Dead yellow dye (Invitrogen) was added to exclude dead cells from analysis. Whenever enough cells were available, corresponding isotype control (Biolegend) staining was also performed. In the event of insufficient cells to run isotype control internal gating controls using markers that provided the cleanest separation between populations were used. Data were acquired on LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo 7.6.4 software (Tree Star, Ashland, OR). Lymphocytes were identified using forward and side scatter, and further gated to include only singlet events and live cells (Figure S1 ). CD19+ cells were subsequently gated to determine the following B cell subsets: CD10+CD27− immature transitional (IT), CD10−CD21+CD27− naïve, CD10−CD21+CD27+ resting memory (RM), CD20+CD21−CD27+ mature activated (MA) and CD10−CD21−CD27− tissue like memory (TLM). PBMC samples with less than 50% viability on trypan blue stain at the time of thaw or live/dead staining were discarded. 5×105–106 total events were captured. In addition there had to be at least 1000 CD19+ cells to analyze the B cell subsets.
3
Comprehensive CAR Expression Analysis
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For evaluation of CAR expression, cells were stained with a goat anti-mouse Fab antibody conjugated with Alexa Fluor 647 (Jackson ImmunoResearch) or biotinylated CD19-Fc (ACRO Biosystems) for 15 min at room temperature. Cells were thoroughly washed before staining with antibodies for additional surface markers. Anti-CD3-BB515 and anti-CD62L-BB515 were purchased from BD Biosciences. Anti-CD4-BV785, anti-CD8-BV711, anti-CD45RA-BV421, anti-CD45RO-PECy7, streptavidin-PE, anti-CD27-PE, anti-CD95-allophycocyanin (APC)Cy7, anti-CCR7-APC, anti-CD45-BV711, and anti-CD19-PE, anti-CD3-BV711 were purchased from BioLegend and used to stain surface antigens. Cell Trace Violet was purchased from Thermo Fisher Scientific and used at a concentration of 1 μM to label cells for 10 min. All data were acquired using a BD Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software.
4
Antigen-experienced B Cell Sorting
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Cryopreserved PBMCs were rapidly thawed in a 37°C water bath for 1 min and then washed with 10 mL cold PBS. After centrifugation, the supernatant was removed, and the PBMCs were resuspended in PBS supplemented with 2% FBS (Cytiva). PBMCs were incubated with Fc-blocker (BioLegend) followed by staining with an antibody cocktail containing anti CD19-AF488 (BioLegend), anti CD3-BV421 (BioLegend), anti CD27-PE (BioLegend), and anti CD38-APC.Cy7 (BioLegend) for 30 min together with fluorescence-labeled antigen probes. Additionally, four different Total-Seq C anti-human hashtag antibodies (BioLegend) were used to label the donor. After three washes, cells from different donors were combined in FACS buffer (2% FBS in PBS) and sorted using BD Aria II Cell Sorter, with the gating strategy described in Fig. S1B. Antigen-experienced B cells (CD19+CD27+) were sorted, and double negative controls were utilized to gate the toxin- and non-binding cells: unlabeled toxin and the non-conjugated anti-His APC (BioLegend) for DT and TT, or non-conjugated Alexa Fluor 647 (Invitrogen) for PT.
5
Characterizing SARS-CoV-2-Specific Memory B Cells
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The SARS-CoV-2-specific MBCs were examined by flow cytometry, as described in a previous study (Chen et al., 2023 (link)). In brief, biotinylated SARS-CoV-2 spike RBD protein (Sino Biological, 40592-V08H2-B) was mixed with streptavidin BV421 (Biolegend, 405225) at a 4:1 molar ratio for 1 hour at 4 °C to obtain the antigen probe. Freshly drawn peripheral blood from participants was processed and stained with specific fluorochrome-conjugated monoclonal antibodies (mAbs), including an antigen probe (1:33.3), anti-CD3-PerCP5.5 (300430, Biolegend, 1:50), anti-CD19-APC (302212, Biolegend, 1:50), anti-CD27-PE (356406, Biolegend, 1:50), and anti-CD21-Alexa Fluor® 700 (354918, Biolegend, 1:50), to perform flow cytometric analysis. Multiparameter flow cytometry detection was performed by CytoFLEX (Beckman Coulter, USA), and the data were analyzed using FlowJo software (10.0.7r2, Treestar).
6
Cytometry and Neutralization Assay for Mycobacterial Antigens
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For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend). For neutralization, we used mAb to IL-4, IL-7, IL-17, IL-21, or isotype-matched control antibody (eBioscience). Recombinant mouse IL-21 was obtained from eBioscience. We used γ-irradiated M. tb H37Rv (γ-M. tb), ESAT6, and Ag85a (all from BEI Resources), and the BCG Tice strain (Organon USA Inc.).
7
Analyzing CD27 Expression in Malignant FL Cells
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To analyse the expression of CD27 in malignant FL B cells and to perform the binding/adhesion assays described below, we used additionally collected cryopreserved FL samples (FL 11–FL 18). Clinical characteristics of patients in the additional FL cohort are described in Supplementary Table 23 . After thawing, cells were filtered through a 70-μm mesh and incubated with PE-anti-CD27 (BioLegend; 1:500), FITC-anti-CD3 (BioLegend; 1:500), APC-anti-CD19 (Miltenyi Biotec; 1:500) and PE-Cy7-anti-CD10 (BioLegend; 1:500) antibodies for 20 min on ice. Cells were then incubated with 7-AAD viability staining solution for 10 min in the dark and analysed using a FACSAria II or III and FlowJo software.
8
Cytometry and Neutralization Assay for Mycobacterial Antigens
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