The design and purification of fluorescently labelled recombinant HA probes with ablated sialic acid binding activity has been previously described (13 (link)). HA-specific B cells were identified within cryopreserved PBMC samples by co-staining with H1 (A/New Caledonia/20/1999) and H5 (A/Indonesia/05/2005) probes conjugated to streptavidin-PE or −APC (Life Technologies, New York, NY) respectively. B cells were stained with the following antibodies for sorting or phenotypic analysis: CD3-QD655, CD14-QD800, CD27-QD605 (Invitrogen), CD19-ECD (Beckman Coulter), IgM-Cy5.5-PerCP, IgG-FITC or −BV421, IgD-Cy7PE, CD38-Alexa Fluor 700, CD22-Cy5PE, CD24-Cy7PE, CXCR5-Ax488, CD20-Cy7APC (BD Pharmingen). Cell viability was assessed using Aqua Live/Dead amine-reactive dye (Invitrogen). The IGHV1-69 anti-idiotypic mouse monoclonal G6 was biotinylated and conjugated to Ax488 using standard procedures. 1 - 2 million events were collected on an LSR II instrument (BD Immunocytometry Systems) configured to detect 18 fluorochromes and analysis was performed using FlowJo software version 9.5.2 (TreeStar). Where applicable, HA probes were incubated with scFv derived from influenza bNAb F10, or alternatively with VRC01 or VRC04 HIV-1 Env-specific scFv controls. Probes were incubated at room temperature with 10 μg/ml of scFv inhibitors for 1h prior to the addition of PBMCs.
Cd38 alexa fluor 700
Manufactured by BD
Sourced in United States
CD38-Alexa Fluor 700 is a fluorescently-labeled antibody that binds to the CD38 cell surface antigen. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 pe, by BD (3 mentions)
Cd19 apc, by BD (3 mentions)
Cd20 apc h7, by BD (3 mentions)
Facsaria 2, by BD (2 mentions)
Live dead blue, by Thermo Fisher Scientific (2 mentions)
Cd14 bv510, by BioLegend (2 mentions)
Cd3 bv510, by BioLegend (2 mentions)
Cd27 bv605, by BioLegend (2 mentions)
Cd56 bv510, by BioLegend (2 mentions)
Cd8 bv510, by BioLegend (2 mentions)
Iga dylight 405, by Jackson ImmunoResearch (2 mentions)
Cd21 bv711, by BD (2 mentions)
Igm brilliant ultraviolet buv 395, by BD (2 mentions)
Igg allophycocyanin apc h7, by BD (2 mentions)
Cd19 bv750, by BioLegend (2 mentions)
Igd pe cy7, by BD (2 mentions)
Facscanto 10, by BD (2 mentions)
Cd10 pe cy7, by BD (2 mentions)
Cd45 percp, by BD (2 mentions)
Syto41, by Thermo Fisher Scientific (2 mentions)
7 protocols using cd38 alexa fluor 700
1
Identification of HA-Specific B Cells
2
Phenotyping PfCSP+ Memory B Cells
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Cryopreserved PBMCs from the VRC 314 trial were thawed and stained for 30 minutes at room temperature in the dark with the following panel: 1:500 Blue LIVE/DEAD (Thermo Fisher Scientific L23105), 18 μg/mL IgA-Dylight 405 (Jackson Immunoresearch 109-475-011), 1:50 CD14-BV510 (BioLegend 301842), 1:50 CD3-BV510 (BioLegend 317332), 1:100 CD8-BV510 (301048), 1:50 CD56-BV510 (BioLegend 318340), 1:50 CD27-BV605 (BioLegend 302830), 1:40 CD21-BV711 (BD Biosciences 563163), 1:50 CD19-BV750 (BioLegend 302236), 1:125 IgD-PECy7 (BD Biosciences 561314), 1:20 IgM-Brilliant Ultraviolet (BUV)395 (BD Biosciences 563903), 1:125 CD38-Alexa Fluor 700 (BD Biosciences 560676), 1:40 IgG-allophycocyanin (APC)H7 (BD Biosciences 561297), 1:25 PfCSP-Brilliant Blue (BB)660, and 1:25 PfCSP-BUV737. The PfCSP probes were made by conjugating biotinylated PfCSP to streptavidin tagged with the relevant fluorescent dye. The cells were sorted using a BD FACS Aria II and analyzed with FlowJo software (Tree Star). Cells were gated on live CD19+CD14-CD3-CD8-CD56-CD21+CD27+IgA+/IgG+, with CD27++CD38++ cells excluded.
3
Multicolor Flow Cytometry for MRD Detection
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Multicolor flow cytometry analysis of BM was performed at the day of diagnosis to assess leukemic-associated immunophenotype (LAIP) of blasts cells. A following antibodies were used for cell immunophenotyping: CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, Waltham, MA, USA), CD58 (Beckman Coulter, Brea, CA, USA). On the day 15th and 33rd BM was analyzed using the same panel of antibodies to determine MRD. To the appropriate amount of BM (106 cells) antibodies listed above were added, samples were incubated for 20 min at room temperature in darkness. Erythrocytes were lysed for 10 min at room temperature in darkness with lysing solution (BD FACS Lysing Solution, Becton Dickinson Biosciences, San Jose, CA, USA), washed twice in PBS, and finally resuspended in 200 μL of PBS. For distinguishing nucleated cells, samples were stained with Syto®41 (Thermo Fisher Scientific, Waltham, MA, USA). FACS analysis was done using FACSCanto or FACSCanto10 with FACSDiva Sorfware v. 8.1 (Becton Dickinson Biosciences, San Jose, CA, USA).
4
Intracellular HO-1 Expression in BM
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HO-1 expression was assessed in BM of 11 patients upon diagnosis. Then in patients in whom, after routine MRD staining, there was a sufficient material for additional staining, an intracellular HO-1 staining was performed (6 children at day 15, 3 children at day 33). In this purpose 106 of bone marrow cells were stained with CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, San Jose, CA, USA), lysed, fixed, and then permeabilized using a BD Intrasure Kit, according to the manufacturer’s instructions. After permeabilization step, cells were incubated with primary antibodies recognizing HO-1 (rabbit polyclonal, SPA 896, Enzo, Warszawa, Poland), washed twice, and stained with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Life technologies, HITACHI, Tokyo, Japan). After two washing steps, samples were stained with Syto®41 (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using FACSCanto10 with FACSDiva Sorfware v 8.0.1 (Becton Dickinson Biosciences, San Jose, CA, USA).
5
B Cell Immunophenotyping by Flow Cytometry
6
Phenotyping PfCSP+ Memory B Cells
7
Immunofluorescence Staining of Hematopoietic Stem Cells
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In order to prepare immunofluorescence slides of certain hematopoietic stem or progenitor cell subsets, characterized elsewhere [68 (link),107 (link)], normal BM samples were stained using following primary antibodies: Lin-FITC, CD90-PE, CD34-APC, CD38-AlexaFluor700, CD45-APC-H7, CD45RA-PE-Cy-7 or CD38-FITC, CD34-PE, CD19-APC, CD20-APC-H7 (BD Biosciences, San Jose, CA, USA). Cells of defined immunophenotype were sorted using MoFlo-XDP cell sorter (Beckman Coulter, Brea, CA, USA) into 20 mL PBS drops on poly-L-lysine coated slides. After settling, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X100. Afterwards, samples were incubated with 0.25% glycine for 30 min, followed by blocking with 3% BSA in PBS for 1 h. Subsequently, samples were stained overnight at 4 °C with primary antibodies recognizing HO-1 (rabbit polyclonal, SPA 896, Enzo, Warszawa, Poland) in a moisture chamber. After 5 washing steps, slides were incubated with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Life technologies, Carlsbad, CA, USA) and DAPI for 1 h in darkness. After the next 5 washing steps, cells were analyzed using a Zeiss confocal microscope with ZEN Software (Zeiss, Oberkochen, Germany).
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