anti cd14 bv510, by BioLegend - Product details

1

Multicolor Flow Cytometry for Kidney Cell Analysis

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An 11-color flow cytometry panel was developed to identify epithelial
cells and leukocyte populations within dissociated kidney cells. Antibodies
include anti-CD45-FITC (HI30), anti-CD19-PE (HIB19), anti-CD11c-PerCP/Cy5.5
(Bu15), anti-CD10-BV421 (HI10A), anti-CD14-BV510 (M5E2), anti-CD3-BV605 (UCHT1),
anti-CD4-BV650 (RPA-T4), anti-CD8-BV711 (SK1), anti-CD31-AlexaFluor700 (WM59),
anti-PD-1-APC (EH12.2H7), and propidium iodide (all from BioLegend). Kidney or
urine cells were incubated with antibodies in HBSS/1%BSA for 30min. Cells were
washed once in HBSS/1%BSA, centrifuged, and passed through a 70 μm
filter. Cells were sorted on a 3-laser BD FACSAria Fusion cell sorter. Intact
cells were gated according to FSC-A and SSC-A. Doublets were excluded by serial
FSC-H/FSC-W and SSC-H/SSC-W gates. Non-viable cells were excluded based on
propidium iodide uptake. Cells were sorted through a 100 micron nozzle at 20
psi. For each sample, 10% of the sample was allocated to sort
CD10⁺CD45⁻ epithelial cells as single cells,
and the remaining 90% of the sample was used to sort CD45⁺ leukocytes
as single cells. Single cells were sorted into 384-well plates containing
6µl 1% NP-40 with index sorting, and plates were immediately frozen and
stored at −80 °C. Flow cytometric quantification of cell
populations was performed using FlowJo 10.0.7.

Arazi A., Rao D.A., Berthier C.C., Davidson A., Liu Y., Hoover P.J., Chicoine A., Eisenhaure T.M., Jonsson A.H., Li S., Lieb D.J., Zhang F., Slowikowski K., Browne E.P., Noma A., Sutherby D., Steelman S., Smilek D.E., Tosta P., Apruzzese W., Massarotti E., Dall’Era M., Park M., Kamen D.L., Furie R.A., Payan-Schober F., Pendergraft WF I.I.I., McInnis E.A., Buyon J.P., Petri M.A., Putterman C., Kalunian K.C., Woodle E.S., Lederer J.A., Hildeman D.A., Nusbaum C., Raychaudhuri S., Kretzler M., Anolik J.H., Brenner M.B., Wofsy D., Hacohen N, & Diamond B. (2019). The immune cell landscape in kidneys of patients with lupus nephritis. Nature immunology, 20(7), 902-914.

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2

Sorting SARS-CoV-2 Spike-Specific B Cells

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Fluorescence-activated cell sorting of cryopreserved PBMCs was performed on a BD FACS Melody. Sorting baits (SARS-CoV-2 Spike) was pre-complexes with the streptavidin flurophore at a 1:4 molar ratio prior to addition to cells. PBMCs were stained with live/dead (fixable Aqua Dead, Thermofisher), anti-CD3-APC/Cy7 (Biolegend), anti-CD8-APC-Cy7 (Biolegend), anti-CD14-BV510 (Biolegend), anti-CD19-PerCP-Cy5.5 (Biolegend), anti-IgM-PE (Biolegend), anti-IgD-Pacific Blue (Biolegend) and anti-IgG-PeCy7 (BD) and Spike-Alexa488 (Thermofisher Scientific, S32354) and Spike-APC (Thermofisher Scientific, S32362). Live CD3/CD8⁻CD14⁻CD19⁺IgM⁻IgD⁻IgG⁺Spike⁺Spike⁺ cells were sorted into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers following the manufacturer’s protocol.

Graham C., Seow J., Huettner I., Khan H., Kouphou N., Acors S., Winstone H., Pickering S., Galao R.P., Lista M.J., Jimenez-Guardeno J.M., Laing A.G., Wu Y., Joseph M., Muir L., Ng W.M., Duyvesteyn H.M., Zhao Y., Bowden T.A., Shankar-Hari M., Rosa A., Cherepanov P., McCoy L.E., Hayday A.C., Neil S.J., Malim M.H, & Doores K.J. (2021). Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike. bioRxiv.

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3

Assessment of NK Cell Activation in HIV

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96-well plates were coated with 1.5μg/ml of gp120 or gp70 V1V2 overnight at 4°C and washed with PBS. Diluted plasma samples were added, incubated at 25°C for 2 hours, and washed with PBS. Cryopreserved PBMCs from one healthy human donor were added at 1 million cells/well and incubated at 37°C for 6 hours in presence of Brefeldin A and Monensin (BD Bioscience).
Cells were surface stained with anti-CD3 AF700 (Clone UCHT1, BD Pharmingen), anti-CD56 PE-Cy7 (Clone B159, BD Biosciences), anti-CD16 APC-Cy7 (Clone 3G8 BD Biosciences), anti-CD19 BV510 (Clone HIB19, BD Biosciences), anti-CD14 BV510 (Clone M5E2, Biolegend) and with Live/dead Fixable Aqua Cell Stain (Thermo Fisher Scientific). Cells were fixed and permeabilized (Cell Fixation & Permeabilization Kit, ThermoFisher) and stained intracellularly for TNF (Clone Mab11, Biolegend), IFNγ (Clone B27, BD Biosciences), and MIP-1β (Clone D21-1351, BD Biosciences). Data were acquired on a BD LSRII instrument and analyzed using FlowJo Version 9.9.6 software. NK cells were gated as follows: singlets (FSC-H v. FSC-A); Aqua-negative; low side scatter; triple negative for CD3, CD19, and CD14; and either CD56+CD16-, CD56+CD16+, or CD56-CD16+ (S9 Fig).

Mdluli T., Jian N., Slike B., Paquin-Proulx D., Donofrio G., Alrubayyi A., Gift S., Grande R., Bryson M., Lee A., Dussupt V., Mendez-Riveria L., Sanders-Buell E., Chenine A.L., Tran U., Li Y., Brown E., Edlefsen P.T., O’Connell R., Gilbert P., Nitayaphan S., Pitisuttihum P., Rerks-Ngarm S., Robb M.L., Gramzinski R., Alter G., Tovanabutra S., Georgiev I.S., Ackerman M.E., Polonis V.R., Vasan S., Michael N.L., Kim J.H., Eller M.A., Krebs S.J, & Rolland M. (2020). RV144 HIV-1 vaccination impacts post-infection antibody responses. PLoS Pathogens, 16(12), e1009101.

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4

Multiparameter Flow Cytometry Characterization

Cited 2 times

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After stimulation, cells were stained for surface and intracellular markers as
performed previously [23 (link), 24 (link)]. The following antibodies and dyes were
used; Aqua LIVE/DEAD (Invitrogen), anti-CD3 BUV396 (UCHT1; BD Biosciences),
anti-CD19 BV510 (SJ25C1; BioLegend), anti-CD14 BV510 (M5E2; BioLegend), anti-CD4
BUV805 (SK3; BD Biosciences), anti-CD8 BV786 (RPA-T8; BD Biosciences), anti-CCR7
PE/Cy7 (G043H7; BioLegend), anti-CD45RA PE/TR (MEM-56; Invitrogen), anti-KLRG1
eFlour710/PerCP (13F12F2; eBioscience), anti-IFN-γ AF700 (B27;
BioLegend), anti-IL-2 APC/Cy7 (MQ1-17H12; BioLegend), anti-TNF-α Pacific
Blue (MAb11; BioLegend), anti-Perforin-1 PE (B-D48; Cell Sciences),
anti-Granzyme B PE/Cy5.5 (GB11; Invitrogen), anti-T-bet BV605 (4B10; BioLegend),
and anti-Eomesodermin (EOMES) eFlour660 (WD1928; eBioscience). Flow cytometric
analysis was performed on a LSRFortessa (BD Biosciences) and analyzed by Flowjo
v10.6.1. Statistics analysis was done by SPICE V6 [25 (link)] and GraphPad Prism 8.

Jin W., Fang M., Sayin I., Smith C., Hunter J.L., Richardson B., Golden J.B., Haley C., Schmader K.E., Betts M.R., Tyring S.K., Cameron C.M., Cameron M.J, & Canaday D.H. (2023). Differential CD4+ T-Cell Cytokine and Cytotoxic Responses Between Reactivation and Latent Phases of Herpes Zoster Infection. Pathogens and Immunity, 7(2), 171-188.

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5

SARS-CoV-2 Antigen-Specific B Cell Sorting

Cited 1 time

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Fluorescence-activated cell sorting of cryopreserved PBMCs was performed on a BD FACS Melody as previously described (Graham et al., 2021 (link)). Sorting baits (SARS-CoV-2 Spike and RBD) was pre-complexed with the streptavidin fluorophore at a 1:4 molar ratio prior to addition to cells. PBMCs were stained with live/dead (fixable Aqua Dead, Thermofisher), anti-CD3-APC/Cy7 (Biolegend), anti-CD8-APC-Cy7 (Biolegend), anti-CD14-BV510 (Biolegend), anti-CD19-PerCP-Cy5.5 (Biolegend), anti-IgM-PE (Biolegend), anti-IgD-Pacific Blue (Biolegend) and anti-IgG-PeCy7 (BD) and Spike-Alexa488 (Thermofisher Scientific, S32354) and Spike-APC (Thermofisher Scientific, S32362) or RBD-Alexa488 and RBD-APC. Live CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺Spike⁺Spike⁺ or CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺RBD⁺RBD⁺ cells were sorted using a BD FACS Melody into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers (Bioline Reagents Ltd) following the manufacturer’s protocol.

Seow J., Graham C., Hallett S.R., Lechmere T., Maguire T.J., Huettner I., Cox D., Khan H., Pickering S., Roberts R., Waters A., Ward C.C., Mant C., Pitcher M.J., Spencer J., Fox J., Malim M.H, & Doores K.J. (2022). ChAdOx1 nCoV-19 vaccine elicits monoclonal antibodies with cross-neutralizing activity against SARS-CoV-2 viral variants. Cell Reports, 39(5), 110757.

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6

Cryopreserved PBMC-based B cell analysis

Cited 1 time

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Cryopreserved PBMC from VRC 018 clinical trial (ClinicalTrials.govNCT03783130)^{22 (link)} at 2 weeks after third boost time point were thawed, treated with Benzonase nuclease (Millipore Corp.), washed with PBS and stained with viability dye Aqua Fluorescent Reactive (Invitrogen) for 2 min followed by staining with the following human antibodies: anti-IgM-BB700 (BD, customized), anti-CD21-PE594 (BD Biosciences, 563474), anti-CD20-APCH7 (BD Biosciences, 560734), anti-IgG-BUV3950 (BD Biosciences, 564229), anti-CD3-BV510 (BD Biosciences, 740187), anti-CD14-BV510 (Biolegend, 301842), anti-CD56-BV510 (BD Biosciences, 740171), anti-CD38-BUV661 (BD Biosciences, 612969), anti-CD19-BUV805(BD Biosciences, 749173), anti-IgD-BV570 (BD Biosciences, 624298), anti-CD27-BV605 (Biolegend, 302830) along with BG505-AF488, glycan-base BG505-PE and glycan-base BG505-APC probes diluted in Brilliant Stain Buffer (BD Biosciences, 563794) for 30 min at 4°C protected from light. Cells were washed with PBS containing 0.1% BSA twice and analyzed on FACS Symphony A5 (BD Biosciences) using Diva software. Cells were gated on live singlets CD3⁻ CD4⁻ CD14⁻ CD56⁻ IgD⁺ IgM⁺ CD19⁺ CD20⁺ IgG⁺ memory B cells and all glycan-base BG505 positive cells were single-cell sorted in 96-well plates coated with 5 μL of TCL buffer (Qiagen) containing 1% of 2-Mercaptoethanol (Sigma-Aldrich).

Arnal C., Ferre-Aubineau V., Mignotte B., Imbert-Marcille B.M, & Billaudel S. (1999). Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA. Applied and Environmental Microbiology, 65(1), 322-326.

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7

Multicolor Flow Cytometry for Kidney Cell Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

An 11-color flow cytometry panel was developed to identify epithelial
cells and leukocyte populations within dissociated kidney cells. Antibodies
include anti-CD45-FITC (HI30), anti-CD19-PE (HIB19), anti-CD11c-PerCP/Cy5.5
(Bu15), anti-CD10-BV421 (HI10A), anti-CD14-BV510 (M5E2), anti-CD3-BV605 (UCHT1),
anti-CD4-BV650 (RPA-T4), anti-CD8-BV711 (SK1), anti-CD31-AlexaFluor700 (WM59),
anti-PD-1-APC (EH12.2H7), and propidium iodide (all from BioLegend). Kidney or
urine cells were incubated with antibodies in HBSS/1%BSA for 30min. Cells were
washed once in HBSS/1%BSA, centrifuged, and passed through a 70 μm
filter. Cells were sorted on a 3-laser BD FACSAria Fusion cell sorter. Intact
cells were gated according to FSC-A and SSC-A. Doublets were excluded by serial
FSC-H/FSC-W and SSC-H/SSC-W gates. Non-viable cells were excluded based on
propidium iodide uptake. Cells were sorted through a 100 micron nozzle at 20
psi. For each sample, 10% of the sample was allocated to sort
CD10⁺CD45⁻ epithelial cells as single cells,
and the remaining 90% of the sample was used to sort CD45⁺ leukocytes
as single cells. Single cells were sorted into 384-well plates containing
6µl 1% NP-40 with index sorting, and plates were immediately frozen and
stored at −80 °C. Flow cytometric quantification of cell
populations was performed using FlowJo 10.0.7.

Arazi A., Rao D.A., Berthier C.C., Davidson A., Liu Y., Hoover P.J., Chicoine A., Eisenhaure T.M., Jonsson A.H., Li S., Lieb D.J., Zhang F., Slowikowski K., Browne E.P., Noma A., Sutherby D., Steelman S., Smilek D.E., Tosta P., Apruzzese W., Massarotti E., Dall’Era M., Park M., Kamen D.L., Furie R.A., Payan-Schober F., Pendergraft WF I.I.I., McInnis E.A., Buyon J.P., Petri M.A., Putterman C., Kalunian K.C., Woodle E.S., Lederer J.A., Hildeman D.A., Nusbaum C., Raychaudhuri S., Kretzler M., Anolik J.H., Brenner M.B., Wofsy D., Hacohen N, & Diamond B. (2019). The immune cell landscape in kidneys of patients with lupus nephritis. Nature immunology, 20(7), 902-914.

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8

Cryopreserved PBMC-based B cell analysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cryopreserved PBMC from VRC 018 clinical trial (ClinicalTrials.govNCT03783130)^{22 (link)} at 2 weeks after third boost time point were thawed, treated with Benzonase nuclease (Millipore Corp.), washed with PBS and stained with viability dye Aqua Fluorescent Reactive (Invitrogen) for 2 min followed by staining with the following human antibodies: anti-IgM-BB700 (BD, customized), anti-CD21-PE594 (BD Biosciences, 563474), anti-CD20-APCH7 (BD Biosciences, 560734), anti-IgG-BUV3950 (BD Biosciences, 564229), anti-CD3-BV510 (BD Biosciences, 740187), anti-CD14-BV510 (Biolegend, 301842), anti-CD56-BV510 (BD Biosciences, 740171), anti-CD38-BUV661 (BD Biosciences, 612969), anti-CD19-BUV805(BD Biosciences, 749173), anti-IgD-BV570 (BD Biosciences, 624298), anti-CD27-BV605 (Biolegend, 302830) along with BG505-AF488, glycan-base BG505-PE and glycan-base BG505-APC probes diluted in Brilliant Stain Buffer (BD Biosciences, 563794) for 30 min at 4°C protected from light. Cells were washed with PBS containing 0.1% BSA twice and analyzed on FACS Symphony A5 (BD Biosciences) using Diva software. Cells were gated on live singlets CD3⁻ CD4⁻ CD14⁻ CD56⁻ IgD⁺ IgM⁺ CD19⁺ CD20⁺ IgG⁺ memory B cells and all glycan-base BG505 positive cells were single-cell sorted in 96-well plates coated with 5 μL of TCL buffer (Qiagen) containing 1% of 2-Mercaptoethanol (Sigma-Aldrich).

Wang S., Matassoli F., Zhang B., Liu T., Shen C.H., Bylund T., Johnston T., Henry A.R., Teng I.T., Tripathi P., Becker J.E., Changela A., Chaudhary R., Cheng C., Gaudinski M., Gorman J., Harris D.R., Lee M., Morano N.C., Novik L., O’Dell S., Olia A.S., Parchment D.K., Rawi R., Roberts-Torres J., Stephens T., Tsybovsky Y., Wang D., Van Wazer D.J., Zhou T., Doria-Rose N.A., Koup R.A., Shapiro L., Douek D.C., McDermott A.B, & Kwong P.D. (2023). HIV-1 neutralizing antibodies elicited in humans by a prefusion-stabilized envelope trimer form a reproducible class targeting fusion peptide. Cell reports, 42(7), 112755.

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Anti cd14 bv510

Lab products found in correlation

8 protocols using anti cd14 bv510

Multicolor Flow Cytometry for Kidney Cell Analysis

Sorting SARS-CoV-2 Spike-Specific B Cells

Assessment of NK Cell Activation in HIV

Multiparameter Flow Cytometry Characterization

SARS-CoV-2 Antigen-Specific B Cell Sorting

Cryopreserved PBMC-based B cell analysis

Multicolor Flow Cytometry for Kidney Cell Analysis

Cryopreserved PBMC-based B cell analysis

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