Peripheral blood monouclear cells (PBMCs) were isolated from patients and healthy individuals, suspended in RPMI 1640 medium containing 2 mM/L L-glutamine and 10% fetal calf serum (Hyclone). Isolated PBMCs were stained with fluorochrome-conjugated antibodies to CD3-PerCP/cy5.5, CD56-FITC, CD8-FITC and CD4-FITC (Biolegend, San Diego, CA). NK cells surface receptors were stained with fluorochrome-conjugated antibodies to NKp46-PE, NKG2A-PE, NKp30-APC, NKG2C-APC, NKG2D-APC and isotype matched controls (Biolegend, San Diego, CA). Stained PBMC were isolated using a FACS Calibur flow cytometer (Becton Dickinson, USA) and analyzed by FlowJo analysis software (Treestar, Ashland, OR).
Nkp30 apc
Manufactured by BioLegend
Sourced in United States
NKp30-APC is a fluorescently labeled antibody that binds to the NKp30 receptor on natural killer cells. NKp30 is an activating receptor involved in the recognition and killing of target cells by natural killer cells. The APC fluorescent label allows for the detection and analysis of NKp30-expressing cells using flow cytometry.
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Lab products found in correlation
Nkg2d apc, by BioLegend (2 mentions)
Dnam 1 fitc, by BioLegend (2 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Cd8 fitc, by BioLegend (1 mentions)
Nkg2a pe, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Nkg2a pe cy7, by Beckman Coulter (1 mentions)
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Cd56 pe cy7, by BioLegend (1 mentions)
Cd45 bv421, by BioLegend (1 mentions)
Cd56 bv510, by BioLegend (1 mentions)
7 aad, by Merck Group (1 mentions)
Nkg2a apc, by Beckman Coulter (1 mentions)
Nkp46 pe, by BioLegend (1 mentions)
Dnam 1 fitc, by BD (1 mentions)
Nkp44 pe, by BioLegend (1 mentions)
Cd16 fitc, by BioLegend (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Nkg2d pe, by BioLegend (1 mentions)
Perforin pe, by BioLegend (1 mentions)
4 protocols using nkp30 apc
1
Immunophenotyping of NK Cell Receptors
2
Phenotyping of HSPC-derived NK Cells
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HSPC-NK cell phenotype was determined by staining for CD56-PE-Cy7 (Biolegend, #318318), NKG2A-APC (Beckman Coulter, #A60797), CD16-FITC (Biolegend, #302006), pan-KIRs-PE (Biolegend, #339506, 312606 and 312708), DNAM-1-FITC (BD, #559788), NKp46-PE (Biolegend, #331908) and NKG2D-APC (Biolegend, #320808) (CellGro vs NK MACS) or CD56-BV510 (Biolegend, #318340), CD45-BV421 (Biolegend, #368522), NKG2A-PE-Cy7 (Beckman Coulter, #B10246), DNAM-1-FITC (Biolegend, #337104), NKp46-PE (Biolegend, #311908), NKp44-PE (Biolegend, #325108), NKp30-APC (Biolegend, #325210) and NKG2D-APC (Biolegend, #320808) (GMP validations runs). Briefly, 200.000 cells were washed using PBS/0.5% BSA and incubated with antibodies in PBS/0.5% BSA at 4 °C for 30 min. Cells were then washed twice with PBS/0.5% BSA and resuspended in PBS/0.5% BSA containing Sytox Blue (1:5000 diluted, invitrogen, #S34857) for CellGro vs NK MACS experiments or 7-AAD (1:1000 diluted, Sigma, #A9400) for GMP validation runs. Cells were acquired on the Gallios (CellGro vs NK MACS) or Navios (GMP validation runs) flowcytometers and analyzed using Kaluza V2.1.3.
3
Comprehensive NK Cell Characterization
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Anti-human CD3-FITC/CD16 + 56-PE mixed antibody were purchased from Beckman Coulter (Brea, CA, USA). Anti-human CD16-FITC, NKG2D-PE, NKp44-APC, DNAM-1-FITC, NKp46-Alexa Fluor 647, NKp30-PE, KIR3DL1-FITC, KIR2DL1/DS1-PE, NKp30-APC, Perforin-PE and Granzyme B-FITC antibodis were all purchased from BioLegend (San Diego, CA, USA), as well as Fixation Buffer, Wash Buffer, Annexin V binding buffer, Alexa Fluor 647 Annexin V and 7-AAD Viability Staining Solution. The MPP-9 and IDO inhibitors were 1-Methyl-DL-tryptopan (1-MT; Sigma-Aldrich, St. Louis, MO, USA) and Tissue inhibitor of metalloproteinases 1 (TIMP-1; PeproTech, Rocky Hill, NJ, USA). Human NK Cell Isolation Kit was purchased from Miltenyi Biotec (Auburn, CA, USA) and ELISA kits were purchased from Abcam (Cambridge, MA, USA). Trizol reagent and PrimeScript RT Master Mix (Perfect Real Time) were both obtained from TaKaRa (Shiga, Japan) and Power SYBR Green PCR Master mix was purchased from Applied Biosystems (Carlsbad, CA, USA).
4
Multiparameter Flow Cytometry Profiling of Immune Cells
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In study 1, 100 μL fresh EDTA blood was stained directly with a pre-prepared and antibody mixture containing: CD3-AlexaFluor700 (Biolegend; clone OKT3), pan-γδTCR-PE (Beckman Coulter; clone IMMU510), CD56-Brilliant Violet(BV)421 (Biolegend; clone HCD56), CD16-APC-eFluor780 (eBiosciences; clone CB16), CD69-PerCP-Cy5.5 (Biolegend; clone FN50), NKp30-APC (Biolegend; clone P30-15), NKG2D-Brilliant Violet(BV)510 (Biolegend; clone 1D11), NKG2A-PEVio770 (Miltenyi Biotec; clone REA110), and CD57-FITC (Biolegend; clone HCD57). A single mixture was prepared one day before the first time point, aliquotted per time point and stored in the dark until use. Samples were stained at 4°C in the dark for 30 min, followed by erythrocyte lysis with 1 mL FACS Lysis buffer (BD Biosciences) for exactly 5 min. Samples were centrifuged and then washed with 0.5% Bovine Serum Albumin (BSA) in PBS. Cell pellets were resuspended in 100 μL 1% paraformaldehyde (PFA) and analyzed on a Gallios flow cytometer (Beckman Coulter). At each time point, staining and fixation was completed within 4 h of blood draw and flow cytometry was performed the same day using identical acquisition settings and a standardized protocol. CD69 was used as a marker for lymphocyte activation after CHMI, as described earlier (20 (link), 21 (link)).
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