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Cd27 qd605

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The CD27-QD605 is a reagent used in flow cytometry applications. It is designed to detect and analyze the expression of the CD27 cell surface marker on cells. The CD27-QD605 utilizes a quantum dot fluorescent label to enable sensitive detection of the target antigen.

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Lab products found in correlation

Cd19 ecd, by Beckman Coulter (4 mentions) Igd cy7pe, by BD (3 mentions) Aqua live dead amine reactive dye, by Thermo Fisher Scientific (2 mentions) Cd8 bv510, by BioLegend (2 mentions) Cd14 bv510, by BioLegend (2 mentions) Facsaria 2, by BD (2 mentions) Live dead aqua, by Thermo Fisher Scientific (2 mentions) Cd56 bv510, by BioLegend (2 mentions) Cd21 cy5pe, by BD (2 mentions) Cd38 alexa fluor 680, by BD (2 mentions) Igm cy5.5 percp, by BD (2 mentions) Igg cy7pe, by BD (2 mentions) Anti cd3 bv510, by BioLegend (2 mentions) Cd20 cy7apc, by BD (1 mentions) Cd3 qd655, by Thermo Fisher Scientific (1 mentions) Cd38 alexa fluor 700, by BD (1 mentions) Lsrii instrument, by BD (1 mentions) F ab 2 fragment goat anti human iga, by Jackson ImmunoResearch (1 mentions) Ctla 4 apc, by Beckman Coulter (1 mentions) Cd127 cy5pe, by BioLegend (1 mentions)

5 protocols using cd27 qd605

1

Identification of HA-Specific B Cells

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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.
Wheatley A.K., Whittle J.R., Lingwood D., Kanekiyo M., Yassine H.M., Ma S.S., Narpala S.R., Prabhakaran M.S., Matus-Nicodemos R.A., Bailer R.T., Nabel G.J., Graham B.S., Ledgerwood J.E., Koup R.A, & McDermott A.B. (2015). H5N1 vaccine-elicited memory B cells are genetically constrained by the IGHV locus in the recognition of a neutralizing epitope in the HA stem. Journal of immunology (Baltimore, Md. : 1950), 195(2), 602-610.
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2

Isolation and Stimulation of Naïve B Cells

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Naïve B cell isolation kit II human (Miltenyi Biotec) was used to isolate naïve B cells from freshly isolated PBMCs according to the manufacturer’s instructions. One hundred thousand naïve B cells were stimulated with WT HA, or Y98F HA, each at 50 nM in RPMI containing 10% FBS. Control cells were stimulated with Anti-Ig [5 μg/ml, F(ab’)2 Fragment Goat Anti-human IgA + IgG + IgM (Jackson)]. After four days, the cells were washed in PBS and resuspended in 50 ul of PBS with Aqua Live/Dead amine-reactive dye (Invitrogen) (0.025 mg/ml for 2 minutes), quenched by addition of 50 ul 1% FBS in PBS, and then stained with a 1X cocktail of following antibodies: CD19 ECD (Beckman), CD27 QD605 (Invitrogen), IgM Cy5PerCP (BD Pharm) for 1 hour at 4 °C. The cells were then washed twice resuspended in 400 μl of PBS and then analyzed by flow cytometry.
Villar R.F., Patel J., Weaver G.C., Kanekiyo M., Wheatley A.K., Yassine H.M., Costello C.E., Chandler K.B., McTamney P.M., Nabel G.J., McDermott A.B., Mascola J.R., Carr S.A, & Lingwood D. (2016). Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation. Scientific Reports, 6, 36298.
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3

Comprehensive Immune Phenotyping by Flow Cytometry

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Flow cytometry was performed using the following directly conjugated antibodies:
(1) BD Biosciences: CD3-H7APC (SK7), CD45RA-Cy7PE (L48), CTLA-4-APC (BNI3),
IFN-g-FITC (B27), CCR7-Alexa700 (150503), CCR5-FITC (2D7/CCR5), CCR4-PE (1G1) and
IL-2-PE (MQ1-17H12), Ki67-FITC (B56); (2) Beckman Coulter: CD27-Alexa680
(IA4CD27), CD127-Cy5PE (R34.34), CD160-PE (BY55), BTLA-PE (J168-540); (3)
BioLegend: PD-1-BV421 (EH12.2H7), 2B4-FITC (CD244, C1.7), IL-17a-Cy5.5PerCP
(BL168), CCR6-Alexa647 (or PE, TG7/CCR6), CD27 Alexa647 (O323), CCR7 BV605
(G043H7), CXCR5 BV421 (J252D4), and CD154-Cy5PE (24–31); (4) Invitrogen:
CD4-Cy5.5PE (S3.5), CD27-QD605 (CLB-27/1), CD8-QD800 (3B5). A biotinylated
anti-PD-1 antibody was obtained from R&D (BAF 1086) and streptavidin-Qdot
655 was obtained from Molecular Probes. Quantum dots and Aqua amine viability dye
were obtained from Invitrogen. CD27-Alexa 594, TNF-a Alexa 594, HLA-DR BV650, and
CD38-Alexa 680 were conjugated in-house.
Paris R.M., Petrovas C., Ferrando-Martinez S., Moysi E., Boswell K.L., Archer E., Yamamoto T., Ambrozak D., Casazza J.P., Haubrich R., Connors M., Ake J., Kim J.H, & Koup R.A. (2015). Selective Loss of Early Differentiated, Highly Functional PD1high CD4 T Cells with HIV Progression. PLoS ONE, 10(12), e0144767.
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4

PfCSP-reactive Memory B Cell Isolation

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PBMCs (106) were stained for viability using the amine-reactive dye Aqua LIVE/DEAD (Invitrogen) followed by staining for surface markers. In addition to the tetramer probes rPfCSP-APC, (NANP)9-PE generated above, the staining panels included: anti-CD3-BV510 (BioLegend), CD8-BV510 (BioLegend), CD14-BV510 (BioLegend), CD56-BV510 (BioLegend), CD19-ECD (Beckman), CD27-QD605 (Invitrogen), CD21-Cy5PE (Becton Dickenson), CD38-Alexa Fluor 680 (Becton Dickenson), IgD-Cy7PE (Becton Dickenson), IgM-Cy5.5-PerCP (Becton Dickenson), and IgG- Cy7PE (Becton Dickenson). Cells were acquired and sorted using a BD FACS Aria II instrument (BD Immunocytometry Systems), and fluorescence-activated cell sorting (FACS) data was analyzed using FlowJo software (Tree Star). Gating strategy is shown in Fig. 1a. PfCSP-reactive (rPfCSP+ and/or (NANP)9+) CD19+CD27+IgG+ IgD−IgM− memory B cells were single cell, dry-sorted into 96-well PCR plates, rapidly frozen on dry ice and stored at −80°C until processing41 (link),42 (link).
Kisalu N.K., Idris A.H., Weidle C., Flores-Garcia Y., Flynn B.J., Sack B.K., Murphy S., Schön A., Freire E., Francica J.R., Miller A.B., Gregory J., March S., Liao H.X., Haynes B.F., Wiehe K., Trama A.M., Saunders K.O., Gladden M.A., Monroe A., Bonsignori M., Kanekiyo M., Wheatley A.K., McDermott A.B., Farney S.K., Chuang G.Y., Zhang B., Natasha K.C., Chakravarty S., Kwong P.D., Sinnis P., Bhatia S.N., Kappe S.H., Sim B.K., Hoffman S.L., Zavala F., Pancera M, & Seder R.A. (2018). A human monoclonal antibody prevents malaria infection and defines a new site of vulnerability on Plasmodium falciparum circumsporozoite protein. Nature medicine, 24(4), 408-416.
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5

PfCSP-reactive Memory B Cell Isolation

Cited 1 time
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PBMCs (106) were stained for viability using the amine-reactive dye Aqua LIVE/DEAD (Invitrogen) followed by staining for surface markers. In addition to the tetramer probes rPfCSP-APC, (NANP)9-PE generated above, the staining panels included: anti-CD3-BV510 (BioLegend), CD8-BV510 (BioLegend), CD14-BV510 (BioLegend), CD56-BV510 (BioLegend), CD19-ECD (Beckman), CD27-QD605 (Invitrogen), CD21-Cy5PE (Becton Dickenson), CD38-Alexa Fluor 680 (Becton Dickenson), IgD-Cy7PE (Becton Dickenson), IgM-Cy5.5-PerCP (Becton Dickenson), and IgG- Cy7PE (Becton Dickenson). Cells were acquired and sorted using a BD FACS Aria II instrument (BD Immunocytometry Systems), and fluorescence-activated cell sorting (FACS) data was analyzed using FlowJo software (Tree Star). Gating strategy is shown in Fig. 1a. PfCSP-reactive (rPfCSP+ and/or (NANP)9+) CD19+CD27+IgG+ IgD−IgM− memory B cells were single cell, dry-sorted into 96-well PCR plates, rapidly frozen on dry ice and stored at −80°C until processing41 (link),42 (link).
Kisalu N.K., Idris A.H., Weidle C., Flores-Garcia Y., Flynn B.J., Sack B.K., Murphy S., Schön A., Freire E., Francica J.R., Miller A.B., Gregory J., March S., Liao H.X., Haynes B.F., Wiehe K., Trama A.M., Saunders K.O., Gladden M.A., Monroe A., Bonsignori M., Kanekiyo M., Wheatley A.K., McDermott A.B., Farney S.K., Chuang G.Y., Zhang B., Natasha K.C., Chakravarty S., Kwong P.D., Sinnis P., Bhatia S.N., Kappe S.H., Sim B.K., Hoffman S.L., Zavala F., Pancera M, & Seder R.A. (2018). A human monoclonal antibody prevents malaria infection and defines a new site of vulnerability on Plasmodium falciparum circumsporozoite protein. Nature medicine, 24(4), 408-416.
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