Flow cytometry antibodies CD62P-APC and PAC1-FITC were from BioLegend (San Diego, CA, USA) and Becton Dickinson (Franklin Lakes, NJ, USA) respectively. Akt S473 (9271), BTK Y551 (18805), DAPP1 Y139 (13703), JAK1 Y1034/1035 (3331), JAK2 Y1007/1008 (3771), JAK3 Y980 (5031), PAK1 S192S197 (2605), PLCγ2 Y1217 (3871), Syk Y525 (2711), STAT3 Y705 (9131), STAT5A/B Y694 (9351), TYK2 Y1054/1055 (9321), phosphorylated Akt substrate (9614) and phosphorylated PKC substrate (6967) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-STAT5a (MAB2174-SP) and STAT5b (MAB15841-SP) were from R&D Systems (Minneapolis, MN, USA). Tubulin (T6199) antibody was from Sigma-Aldrich. Alexa Fluor 488 goat anti-mouse (A21042) and 594 goat anti-rabbit A32740) secondary antibodies were from Invitrogen (ThermoFisher Scientific; Carlsbad, CA, USA).
Pac1 fitc
Manufactured by BioLegend
Sourced in United States
PAC1-FITC is a fluorochrome-conjugated antibody that binds to the PAC1 (Platelet Activation Complex-1) receptor on the surface of activated platelets. It can be used to detect and quantify platelet activation in flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cd62p apc, by BioLegend (2 mentions)
Phosphorylated pkc substrate 6967, by Cell Signaling Technology (2 mentions)
Tubulin t6199 antibody, by Merck Group (2 mentions)
Cd62 pe, by BioLegend (2 mentions)
Fitc mouse igm k isotype, by BioLegend (2 mentions)
Igg1 k, by BioLegend (2 mentions)
Cd41 pe cy7, by BioLegend (2 mentions)
Mouse igg1 k isotype, by BioLegend (2 mentions)
Cd8 apc, by BioLegend (1 mentions)
Cd4 pe cy7, by BioLegend (1 mentions)
Annexin 5 fitc 7 aad, by BioLegend (1 mentions)
Cd19 pe, by BioLegend (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
5 protocols using pac1 fitc
1
Flow Cytometry-Based Signaling Pathway Analysis
2
Platelet and PBMC Evaluation Protocol
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For the evaluation of PAC‐1 and CD62P on platelets, anticoagulated whole blood was immediately stained (referred as resting platelets) or stimulated by thrombin receptor activating peptide (TRAP, Merck KGaA, Darmstadt, Germany) for 20 min (referred as platelets with TRAP) and then stained with the following anti‐human antibodies (Biolegend, San Diego, USA): CD61‐Percp/Cy5.5, CD62P‐PE and PAC‐1‐FITC. This procedure was gently operated at room temperature and completed within 1 h after blood withdrawal.
For the apoptosis of PBMC, cells were collected and stained with the following anti‐human antibodies or fluorescent dye (Biolegend, San Diego, USA) according to the manufacturer's instructions: Annexin V‐FITC, 7‐AAD, CD19‐PE, CD4‐PE/CY7, CD8‐APC, CD14‐ APC/Cyanine7 and CD56‐Brilliant Violet 510. The acquisition was performed on a FACS AriIII flow cytometer (BD Biosciences, New York, USA) and then analysed using Flowjo software version 10.0.1. Annexin V+ 7‐AAD− cells were considered as apoptotic cells.
For the apoptosis of PBMC, cells were collected and stained with the following anti‐human antibodies or fluorescent dye (Biolegend, San Diego, USA) according to the manufacturer's instructions: Annexin V‐FITC, 7‐AAD, CD19‐PE, CD4‐PE/CY7, CD8‐APC, CD14‐ APC/Cyanine7 and CD56‐Brilliant Violet 510. The acquisition was performed on a FACS AriIII flow cytometer (BD Biosciences, New York, USA) and then analysed using Flowjo software version 10.0.1. Annexin V+ 7‐AAD− cells were considered as apoptotic cells.
3
Flow Cytometry Antibodies and Signaling Assays
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Flow cytometry antibodies CD62P-APC and PAC1-FITC were from BioLegend (San Diego, CA, USA) and Becton Dickinson (Franklin Lakes, NJ, USA) respectively. TLT1 was from R&D Systems (#FAB2394G). Akt S473 (9271), NF-κB p65 S536 (3033), BTK Y551 (18805), DAPP1 Y139 (13703), PLCγ2 Y1217 (3871), Syk Y525 (2711), phosphorylated Akt substrate (9614), phosphorylated MAPK substrate (2325), and phosphorylated PKC substrate (6967) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). 4G10 was from Sigma Millipore (#05-321). Tubulin (#T6199) antibody was from Sigma-Aldrich. Anti CD41-FITC antibody was from BD Biosciences (#555466).
4
Platelet Activation Markers Evaluation
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CD41/PECy7 (BioLegend Cat. No. 303718) was used as an identity marker for platelets, PAC-1/FITC (BioLegend Cat. No. 362804) for glycoprotein GP IIbIIIa and CD62/PE (BioLegend Cat. No. 304906) for P-selectin were used as activation markers. IgG1 k (BioLegend Cat. No. 400125), FITC Mouse IgM k Isotype (BioLegend Cat. No. 401605) and Mouse IgG1 k Isotype (BioLegend Cat. No. 400111) were used as isotype control, respectively. The gating strategy of the cell populations was performed according to previously reported by the research group in García-Larragoiti et al. [22 (link)]. Dark conditions and minimal handling were used during the assay to avoid external activation of platelets. Adenosine Di Phosphate (ADP) (20 µM) for 20 min, collagen (20 µM) for 30 min, and epinephrine (EPI) (100 µM) for 40 min were used as positive platelet activation controls [27 (link)]. Concentrations were used following the instructions suggested by the supplier PAR/PAK II® BIO/DATA CORPORATION (Horsham, PA, USA). The acquisition was performed in a CytoFLEX, BECKMAN COULTER® (Brea, CA, USA). Results were analyzed using FlowJo v 10.8.0.
5
Platelet Activation Assay by Flow Cytometry
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PRP was obtained by centrifugation, diluted in Tyrodes buffer and incubated for
20 min with CD41/PECy7 (BioLegend Cat. No. 303718) as identity marker,
PAC-1/FITC (BioLegend Cat. No. 362804) and CD62/PE (BioLegend Cat. No. 304906),
were both used as activation markers (glycoprotein αIIbβIII and P-selectin,
respectively). IgG1 k (BioLegend Cat. No. 400125), FITC Mouse IgM k Isotype
(BioLegend Cat. No. 401605) and, Mouse IgG1 k Isotype (BioLegend Cat. No.
400111) were used as isotype control respectively. Subsequently, platelets were
fixed with paraformaldehyde at 4%. Dark conditions and minimal handling were
used during the assay to avoid external activation of platelets. As positive
controls of platelet activation we used known activation agonists ADP, collagen
and epinephrine and the acquisition was performed by flow cytometry as reported
before by our group.10 (link) Results were analyzed using FlowJo v 10.8.0.
20 min with CD41/PECy7 (BioLegend Cat. No. 303718) as identity marker,
PAC-1/FITC (BioLegend Cat. No. 362804) and CD62/PE (BioLegend Cat. No. 304906),
were both used as activation markers (glycoprotein αIIbβIII and P-selectin,
respectively). IgG1 k (BioLegend Cat. No. 400125), FITC Mouse IgM k Isotype
(BioLegend Cat. No. 401605) and, Mouse IgG1 k Isotype (BioLegend Cat. No.
400111) were used as isotype control respectively. Subsequently, platelets were
fixed with paraformaldehyde at 4%. Dark conditions and minimal handling were
used during the assay to avoid external activation of platelets. As positive
controls of platelet activation we used known activation agonists ADP, collagen
and epinephrine and the acquisition was performed by flow cytometry as reported
before by our group.10 (link) Results were analyzed using FlowJo v 10.8.0.
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