3T3 and 3T3hEGFR cells (0.3 × 105 cells/well were pre-coated in 96-well plates overnight at 37°C. Next day, target cells were pre-incubated for 1 h at 37°C with purified antibodies (6.67 nM) and a mouse IgG1K isotype (Biolegend), as a control. Mouse CD8a+ T cells were purified from the spleens of female BALB/c mice using the mouse CD8a+ T cell Isolation Kit (Miltenyi Biotec). Purified mouse CD8a+ T cells (1 × 105 cells/well) were resuspended in RPMI supplemented with 10% FBS and 50 μM β-mercaptoethanol (Life Technologies), activated with anti-CD3 mAb (clone 145-2C11; MO3PU, Immuno-step) (1 μg/mL) and added to the coculture. As a control, mouse CD8a+T cells were cultured alone with or without anti-CD3 mAb (1 μg/mL). After 72 h supernatants were collected and assayed for IFN-γ secretion by ELISA (Diaclone) following manufacturer’s protocol.
Mouse igg1 k isotype
Manufactured by BioLegend
Mouse IgG1 κ Isotype is a laboratory product used as a control antibody in various immunoassays and flow cytometry applications. It serves as a reference to establish baseline or non-specific binding levels when analyzing samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd62 pe, by BioLegend (2 mentions)
Fitc mouse igm k isotype, by BioLegend (2 mentions)
Igg1 k, by BioLegend (2 mentions)
Pac1 fitc, by BioLegend (2 mentions)
Cd41 pe cy7, by BioLegend (2 mentions)
Mouse cd8a t cell isolation kit, by Miltenyi Biotec (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Cd24 fitc, by BD (1 mentions)
Cd162 alexa fluor 647, by BD (1 mentions)
Anti p selectin, by BioLegend (1 mentions)
Alexa fluor 488, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
4 protocols using mouse igg1 k isotype
1
Antibody-mediated CD8+ T cell activation
2
Flow Cytometry Analysis of HGSOC Cells
3
Platelet Activation Markers Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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.
4
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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