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Cd158b fitc

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CD158b-FITC is a fluorochrome-conjugated monoclonal antibody that binds to the CD158b antigen, also known as killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1). It is used in flow cytometry applications to identify and analyze KIR3DL1-positive cells.

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Lab products found in correlation

Fixable blue dead cell stain, by Thermo Fisher Scientific (2 mentions) Kir3dl1 alexafluor700, by BioLegend (2 mentions) Nkg2a pe, by Beckman Coulter (2 mentions) Cd94 fitc, by BD (2 mentions) 2b4 fitc, by BD (2 mentions) Nkp46 pe, by BD (2 mentions) Nkp30 apc, by BD (2 mentions) Trail pe, by BioLegend (2 mentions) Cd161 fitc, by Miltenyi Biotec (2 mentions) Cd160 alexafluor647, by BioLegend (2 mentions) Cd158a percp cy5, by Thermo Fisher Scientific (2 mentions) Anti perforin percp cy5, by Thermo Fisher Scientific (2 mentions) Nkg2d apc, by BD (2 mentions) Cd56 pe cy7, by BioLegend (1 mentions) Apc cy7, by BioLegend (1 mentions) Cd3 ecd, by Beckman Coulter (1 mentions) Cd158e1 bv421, by BioLegend (1 mentions) Tnf α af647, by BioLegend (1 mentions) Cd107a fitc, by BD (1 mentions) Lsrii flow cytometer, by BD (1 mentions)

6 protocols using cd158b fitc

1

Flow Cytometric Analysis of NK Cell Receptors

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The antibodies used in this study were CD56 PE-Cy7 and APC-Cy7, CD158a/CD158b/CD158e1 PE (used in experiments were KIR were pooled), CD158e1 BV421, TNF-α AF647, IFN-γ Pacific blue, DLL1 and DLL4 PE, purified mouse IgM isotype control (BioLegend), CD158b FITC, CD107a FITC, purified mouse anti-human CD16 (BD Bioscience), CD3 ECD, CD158b APC (Beckman Coulter), and CD158a PE-Cy7 (eBioscience). For CD16 activation studies, cells were cultured with anti-CD16 or isotype control for 30 min and then crosslinked with goat-anti mouse IgG for 5 hours. Staining, acquisition, and analysis were performed as previously described (27 (link)). Finally, cells were run on an LSRII flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (TreeStar).
Felices M., Ankarlo D.E., Lenvik T.R., Nelson H.H., Blazar B.R., Verneris M.R, & Miller J.S. (2014). Notch signaling at later stages of Natural Killer cell development enhances KIR expression and functional maturation. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3344-3354.
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2

Comprehensive NK-Cell Receptor Profiling

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Cryopreserved peripheral blood mononuclear cells (PBMC) were stained with combinations of antibodies that comprehensively interrogate the breadth of NK-cell receptors including the killer immunoglobulin receptors (KIRs), the c-type lectin receptors (NKG2), and the natural cytotoxicity receptors (NCRs). These included CD158a-PerCP-Cy5.5 (eBioscience), KIR3DL1-Alexafluor700 (Biolegend), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC, CD158b-FITC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). Dead cells were excluded using the Fixable Blue Dead Cell Stain (Life Technologies) prior to surface staining and fixation. For perforin staining, samples were then permeabilized (Perm A/B, Caltag) and stained with anti-Perforin-PerCP-Cy5.5 (eBioscience). NK cells were identified as CD3 negative lymphocytes expressing CD56 and/or CD16. At least 1500 NK cells were acquired per sample. Fluorescence minus one was used to set gates. The data were analyzed with Flowjo v9.5.4 (Treestar).
Yu W.H., Cosgrove C., Berger C.T., Cheney P.C., Krykbaeva M., Kim A.Y., Lewis-Ximenez L., Lauer G.M, & Alter G. (2018). ADCC-Mediated CD56DIM NK Cell Responses Are Associated with Early HBsAg Clearance in Acute HBV Infection. Pathogens & Immunity, 3(1), 2-18.
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3

Multiparametric Flow Cytometry Analysis

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For phenotype analysis, cells were stained with 7AAD (Beckman) to identify viable cells and antibodies against the surface markers CD25-FITC, CD45RO-FITC, CD69-PE, CD62L-PE, CD19-PE, CD3-PE, CD19-ECD, CD56-PECy7, CD56-APC, CD3-APC, CD45-APCAlexaFluor750, CD45RA-APCAlexaFluor750, CD16-PacificBlue, CD57-PacificBlue, CD45-KromeOrange, CD16-KromeOrange (Beckman), CD158b-FITC, CD158a-PE, CD107a-HV500 (BD Biosciences), CD158e-Vioblue (Miltenyi). 1×105-3×105 cells were incubated for 20-30 min at 4 °C with different antibodies in PBS containing 2.5% FBS. Cells were then washed and suspended in 200-250 µL of the same media. Staining was analyzed on a Gallios flow cytometer (Beckman) using the Kaluza software. Alive lymphocytes were gated using FSC/SSC and 7AAD staining. B lymphocytes (CD19+), T lymphocytes (CD3+CD56-) and NK cells (CD56+CD3-) were distinguished using, respectively, CD19, CD3 and CD56 antibodies.
Sanchez-Martinez D., Allende-Vega N., Orecchioni S., Talarico G., Cornillon A., Vo D.N., Rene C., Lu Z.Y., Krzywinska E., Anel A., Galvez E.M., Pardo J., Robert B., Martineau P., Hicheri Y., Bertolini F., Cartron G, & Villalba M. (2018). Expansion of allogeneic NK cells with efficient antibody-dependent cell cytotoxicity against multiple tumors. Theranostics, 8(14), 3856-3869.
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4

Comprehensive Immune Cell Profiling

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The following mAbs were used to detect surface markers: anti-CD3 APC-Cy7 (clone: UCHT1), CD14 APC-Cy7 (HCD14), CD16 BV510 (3G8), CD19 APC-Cy7 (HIB19), CD25 BV421 (BC96), CD69 BV421 (FN50), CD56 PE-Cy7 (HCD56), CD4 FITC (RPAT4) and CD62L (DREG-56), all IgG1 isotypes from Biolegend. CD57 VioBlue (TB03. isotype IgM) CD159C PE (REA 205, isotype IgG1), CD279 PE (PD1.3.1.3. isotype: IgG2B), CD8 Vioblue (REA734 isotype IgG1) and CD45RA PE-Vio770 (REA 562. isotype IgG1) were from Miltenyi Biotech Bergisch, Gladbach, Germany, CD158a FITC (HP-3E4 isotype IgM), CD158b FITC (CH-L. isotype IgG2b) and CD27 BV510 (L128. isotype IgG1) were from BD Biosciences. CA. USA. CD159a APC (Z199. isotype IgG2b) and CD336 PE (Z231. Isotype IgG1) were from Beckman Coulter, (Beckman Coulter. 250 S. Kraemer Blvd, Brea, CA 92821, USA).
Bergantini L., d’Alessandro M., Cameli P., Cavallaro D., Gangi S., Cekorja B., Sestini P, & Bargagli E. (2021). NK and T Cell Immunological Signatures in Hospitalized Patients with COVID-19. Cells, 10(11), 3182.
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5

Characterization of NK Cell Receptors

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PBMC were obtained from buffy coats of healthy donors (n=4) by Ficoll-Paque™ PLUS Media gradient centrifugation (Thermo Fisher Scientific, USA). PBMC were treated as described above and the expression of activating and inhibitory receptors on NK cells was determined by flow cytometry on day 3 and 7 using following fluorescently labeled antibodies in two panels: CD45-PE-Dy594 (Exbio, Czech Republic), CD3-APC-eFluor780 (eBioscience, USA), NKG2D-PE-Cy7, CD56-Alexa Fluor700, CD16-PE-Cy7, DNAM-1-FITC (all from Biolegend, USA), NKp30-PE, NKG2A-APC (R&D), CD158a-PE and CD158b-FITC (BD Biosciences, USA). The dead cells detected by LIVE/DEAD Fixable Aqua stain (Thermo Fisher Scientific, USA) were excluded from the analyses. The gating strategy was as follows: singlets/live CD45+/CD3-/CD56+/receptor+. Mean fluorescence intensity (MFI) of receptor expression on NK cells was determined from CD3-CD56+ cell population.
Antosova Z., Podzimkova N., Tomala J., Augustynkova K., Sajnerova K., Nedvedova E., Sirova M., de Martynoff G., Bechard D., Moebius U., Kovar M., Spisek R, & Adkins I. (2022). SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity. Frontiers in Immunology, 13, 989895.
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6

Comprehensive NK-Cell Receptor Profiling

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Cryopreserved peripheral blood mononuclear cells (PBMC) were stained with combinations of antibodies that comprehensively interrogate the breadth of NK-cell receptors including the killer immunoglobulin receptors (KIRs), the c-type lectin receptors (NKG2), and the natural cytotoxicity receptors (NCRs). These included CD158a-PerCP-Cy5.5 (eBioscience), KIR3DL1-Alexafluor700 (Biolegend), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC, CD158b-FITC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). Dead cells were excluded using the Fixable Blue Dead Cell Stain (Life Technologies) prior to surface staining and fixation. For perforin staining, samples were then permeabilized (Perm A/B, Caltag) and stained with anti-Perforin-PerCP-Cy5.5 (eBioscience). NK cells were identified as CD3 negative lymphocytes expressing CD56 and/or CD16. At least 1500 NK cells were acquired per sample. Fluorescence minus one was used to set gates. The data were analyzed with Flowjo v9.5.4 (Treestar).
Yu W.H., Cosgrove C., Berger C.T., Cheney P.C., Krykbaeva M., Kim A.Y., Lewis-Ximenez L., Lauer G.M, & Alter G. (2018). ADCC-Mediated CD56DIM NK Cell Responses Are Associated with Early HBsAg Clearance in Acute HBV Infection. Pathogens & Immunity, 3(1), 2-18.
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