To determine IGF1R expression, 200 μL of whole blood was stained with the following fluorescently-labeled anti-human antibodies for thirty minutes at 23°C in the dark: CD25-AF488 (BC96), CD127-BV421 (A019D5), CD197-AF647 (G043H7), CD45RA-PerCP-Cy5.5 (HI100), CD3-APC-Fire750 (SK7), CD221-PE (1H7) (BioLegend), CD4-PE-Cy7 (RPA-T4, eBioscience). RBCs were lysed for five minutes at 23°C with 1-step Fix/Lyse Solution (eBioscience), followed by three washes with stain buffer. Data were acquired on an LSRFortessa (BD Biosciences) and analyzed with FlowJo software (v10.6.1; Tree Star).
Cd25 af488
Manufactured by BioLegend
CD25-AF488 is a fluorescently-labeled anti-CD25 antibody. CD25 is the alpha chain of the IL-2 receptor and is expressed on activated T cells, B cells, and NK cells. The antibody is conjugated to the Alexa Fluor 488 fluorescent dye, which can be detected using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
1 step fix lyse solution, by Thermo Fisher Scientific (1 mentions)
Cd45ra percp cy5, by BioLegend (1 mentions)
Cd127 bv421, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cd3 af488, by BioLegend (1 mentions)
Cd45 af488, by BioLegend (1 mentions)
Cd14 af488, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
2 protocols using cd25 af488
1
Quantifying IGF1R Expression in Whole Blood
2
Flow Cytometry Analysis of Activated Immune Cells
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The activated/rested cells were plated into a 96-well round bottom plate and pelleted at low speed. Cells were resuspended in chilled media containing titrated HS-131 or HS-198 (in 1% DMSO) and stained for 30 min at RT. Fluorochrome tagged inhibitors and media were washed away with a PBS wash, followed by a PBS wash containing 3% NMS. Surface antibody stains were performed in 3% NMS/PBS for 30 min followed by PBS washes (2×) (Table 2 ). Samples were analyzed on BD FACSCanto II flow cytometer. Permeabilized samples were prepared as previously described followed by fixation in 4% Formaldehyde/PBS overnight at 4 °C. Minimum gating of 2X105 cells/sample. Sample analysis was performed with Flowing Software and FlowJo.
Flow cytometry antibodies.
| Antibody | Target and fluor | Manufacturer | Concentration |
|---|---|---|---|
| 300434 | CD3- BV421 | BioLegend | 1:200 |
| 302616 | CD25-AF488 | BioLegend | 1:200 |
| 423113 | Viability-BV421 | BioLegend | 1:1000 |
| 423102 | Viability-BV510 | BioLegend | 1:1000 |
| 310903 | CD69-FITC | BioLegend | 1:200 |
| 302605 | CD25-PE | BioLegend | 1:200 |
| 561105 | Ly6G-AF488 | BD Biosciences | 1:500 |
| 103139 | CD45-BV605 | BioLegend | 1:800 |
| 563415 | IA/IE-BV650 | BD Biosciences | 1:1500 |
| 103121 | CD45-AF488 | BioLegend | 1:1000 |
| 300454 | CD3-AF488 | BioLegend | 1:1000 |
| 301817 | CD14-AF488 | BioLegend | 1:1000 |
| 400625 | Rat IgG2b Isotype Control | BioLegend | 1:1000 |
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