Cd158a

Manufactured by BD

CD158a is a receptor molecule expressed on the surface of certain immune cells. It plays a role in the regulation of immune responses, but its precise function requires further research.

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3 protocols using cd158a

Quantification of NK Cell Subsets

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PBMCs were washed in cold PBS with 2% FCS and 0.1% sodium azide and then stained with fluorescein isothiocyanate (FITC)-, R-phycoerythrin (PE) or Allophycocyanin (APC)-conjugated mouse anti-human monoclonal antibodies including anti-CD3/CD16⁺CD56 (FITC/APC), CD69, CD94, NKAT-2, NKG2D, NKp46, NKp30, NKG2A,CD158a, CD158b, and CD158k (PE) (BD Biosciences) from Becton-Dickinson (Worldwide Inc., Taiwan Branch, Taiwan) for flow cytometric analysis.
The fluorescent staining was analyzed on a FACS Calibur (BD Biosciences) flow cytometer. Electronic gates were set to enable analysis of the fluorescence of the viable cell population according to FSC/SSC histograms following anti-CD3/ CD16+CD56 staining. The percentage of cells stained with each monoclonal antibody was determined by comparing each histogram with one from control cells stained with FITC-, PE or APC-labeled isotype control monoclonal antibodies. The lymphocyte population was gated first to identify CD3-positive and CD3-negative lymphocyte populations for the subsequent analysis of the expression patterns of CD56. According to the mean fluorescence intensity of CD56, CD56⁺CD3⁺ NK cells were further divided by fluorescent intensity of CD56 staining in to 2 groups: CD56^dim (more than 80%) NK cells and CD56^bright NK cells.

Lin S.J., Kuo M.L., Hsiao H.S., Lee P.T., Chen J.Y, & Huang J.L. (2017). Activating and inhibitory receptors on natural killer cells in patients with systemic lupus erythematosis-regulation with interleukin-15. PLoS ONE, 12(10), e0186223.

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Phenotyping of NK Cells by Flow Cytometry

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Fluorochrome‐labeled monoclonal antibodies specific for CD3 (V500) and CD56 (V450) (BD Biosciences, San Jose, CA) were used to identify NK (CD3^negCD56^pos) cells within the overall lymphocyte population. Anti‐NKR antibodies CD16, CD161, CD122, CD158a, CD158e, CD94, CD69, CD95 (FAS), CD226 (DNAM), CD253 (TRAIL), and CD314 (NKG2D) were purchased from BD Biosciences. Anti‐CD335 (NKp46), CD337 (NKp30), and CD159a (NKG2A) were purchased from Beckman Coulter (Indianapolis, IN). Anti‐CD158b and CD328 (Siglec‐7) were purchased from eBioscience (San Diego, CA). Anti‐Tim‐3 was purchased from R&D Systems (Minneapolis, MN).
Thawed PBMCs (1‐2 × 10⁶) were stained for cell‐surface antigen expression at 4°C in the dark for 30 minutes. Samples were then washed twice in 2 mL phosphate‐buffered saline containing 1% bovine serum albumin and 0.01% sodium azide (Facs Wash; MilliporeSigma, St. Louis, MO) and subsequently fixed in 200 μL 1X BD stabilization fixative. Isotype‐matched control antibodies were used to determine background levels of staining. Multicolor flow cytometry was performed using a BD FACSCanto II instrument (BD Biosciences), compensated with single fluorochromes and analyzed using Diva software (BD Biosciences). Lymphocyte populations were identified by their characteristic forward scatter/side scatter properties.

Golden‐Mason L., McMahan R.H., Kriss M.S., Kilgore A.L., Cheng L., Dran R.J., Wieland A, & Rosen H.R. (2018). Early and late changes in natural killer cells in response to ledipasvir/sofosbuvir treatment. Hepatology Communications, 2(4), 364-375.

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Phenotypic Profiling of NK Cells

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The following antibodies were used: CD34 (PercpCy5.5 or PE, clone 581), CD56 (PercpCy5.5 or PeCy7, clone B159), CD94 (FITC, clone HP-3D9), CD117 (PeCy7, clone 104D2 or PercpCy5.5, clone YB5.B8), CD161 (FITC,clone DX12), CD14 (ApcCy-7, clone MφP9), CD158a (FITC clone, HP3E4), CD158b (FITC, clone CH-2), NKB1 (FITC, clone DX9) all from BD Biosciences, San Jose, CA. Additional antibodies included NKG2D (PE, clone 149810), NKp30 (PE, clone 210845), NKp44 (PE, clone 253415), and NKp46 (PE, clone 195314) all obtained from R&D Systems, Minneapolis, MN. Intracellular staining for IFN-γ (PE clone 45.B3) was performed using cytofix/cytoperm (BD Biosciences). IFN-γ staining was performed after 16 hours of stimulation with IL-12 (10 ng/mL) and IL-18 (100 ng/mL). Brefeldin A was added for the last 4 hours. Data were analyzed using Flowjo Version 7.6. FACS sorting was performed on either a FACS Vantage or FACS Aria (BD Biosciences).

Weeres M.A., Robien K., Ahn Y.O., Neulen M.L., Bergerson R., Miller J.S, & Verneris M.R. (2014). The Effects of 1,25 Dihydroxyvitamin D3 (1,25(OH)2D3) on In Vitro Human Natural Killer Cell DevelopmentFrom Hematopoietic Stem Cells. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3456-3462.

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