For intracellular staining of phosphor-proteins, cells were fixed with 2% paraformaldehyde, followed by permeabilization in 95% methanol. The following antibodies were used: anti-p-PDHE1α (AP1062; Calbiochem, Darmstadt, Germany) and a fluorescein isothiocyanate conjugated antirabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX). For staining of intracellular cytokines/transcription factors, either the Mouse Foxp3 Buffer Set 560409 (BD Biosciences, Franklin Lakes, NJ) or the Foxp3/Transcription Factor Staining Buffer Set 00-5523-00 (eBioscience, San Diego, CA) was used along with the following antibodies: CD45-BV421, CD3-PECy7, CD4-APC, CD8-Alexafluor700, NK1.1-BV711, IFN-γ-BV510, IL-4-BV605, IL-17-PerCP-Cy5.5, CD25-BV785, Foxp3-FITC, CD69-PerCPCy5.5, and CD62L-PerCPCy5.5 (all from BioLegend, San Diego, CA). Cell viability was measured using a Live/Dead staining kit (Thermo Fisher). Cell death was measured using an FITC/Annexin V Apoptosis Detection Kit (556547; BD Biosciences).
Cd69 percpcy5
Manufactured by BioLegend
CD69-PerCPCy5.5 is a fluorescently labeled antibody that binds to the CD69 antigen, which is a type II transmembrane protein expressed on the surface of activated T cells, B cells, and natural killer cells. The antibody is conjugated with the PerCPCy5.5 fluorescent dye, which can be detected using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 bv421, by BioLegend (1 mentions)
Cd8 alexa fluor 700, by BioLegend (1 mentions)
Nk1.1 bv711, by BioLegend (1 mentions)
Ifn γ bv510, by BioLegend (1 mentions)
Il 4 bv605, by BioLegend (1 mentions)
Il 17 percp cy5, by BioLegend (1 mentions)
Live dead staining kit, by Thermo Fisher Scientific (1 mentions)
Foxp3 fitc, by BioLegend (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Cd62l percp cy5, by BioLegend (1 mentions)
Cd3 pe cy7, by BioLegend (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cd3 alexa 488, by BioLegend (1 mentions)
Ready set go elisa kit, by Thermo Fisher Scientific (1 mentions)
Cd86 percp cy5, by BioLegend (1 mentions)
Anti cd11c alexa488, by BioLegend (1 mentions)
Cd19 alexa647, by BioLegend (1 mentions)
Cd80 alexa647, by BioLegend (1 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
Cd27 biotin, by BioLegend (1 mentions)
4 protocols using cd69 percpcy5
1
Intracellular Staining and Cytokine Detection
2
Immunomodulatory Effects of GD5 and DOX
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One week after the tumor injection, the tumor-bearing mice were treated intraperitoneally with 2.5 mg/kg GD5 or 3.5 mg/kg DOX. After 2 and 4 h, the spleens were collected, and the splenocytes were prepared according to the manufacturer's instructions (BioLegend). Approximately 1 × 106 cells were stained with surface antibodies and analyzed by FACSCalibur flow cytometry (BD Biosciences). The antibodies including anti-CD11c-Alexa488, -CD86-PerCP/Cy5.5, -CD80-Alexa647, -CD3-Alexa488, -CD19-Alexa647 and -CD69-PerCP/Cy5.5 were purchased from BioLegend.
Sera samples were collected at 2, 4 or 24 h from the same administrated mice and stored at -20°C. ELISA was performed using a Ready-SET-Go!® ELISA kit (eBioscience).
Sera samples were collected at 2, 4 or 24 h from the same administrated mice and stored at -20°C. ELISA was performed using a Ready-SET-Go!® ELISA kit (eBioscience).
3
Comprehensive Murine NK Cell Immunophenotyping
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The following antibodies were purchased from Biolegend: CD3 BV510, CD49b (DX5) APC-Cy7, NKp46 BV421, CD11b PE, Ly49D PE, Streptavidin BV650, CD27 biotin, KLRG1 BV605, KLRG1 PerCP/Cy5.5, CD69 PerCP/Cy5.5, and Ki67 APC. The following antibodies were purchased from eBioscience: CD107a PerCP-eFluor710, Ly49I PE, CD94 FITC, IFNγ APC, and Ly49H PE-Cyanine7. The following antibodies were purchased from BD Biosciences: NKG2A/C/E BV605. The following antibodies were purchased from Miltenyl Biotec: NKG2D (CD314) biotin, Ly49D biotin, and Ly49D PerCP-Vio700. To determine viability Invitrogen Fixable live/dead Aqua stain was used.
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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