Identifying Kinase Inhibitors that Modulate the Thymocyte Response to Strong TCR Signals
Published: August 31, 2023
Abstract
Source: Chen, E. W., et al. Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries. J. Vis. Exp. (2019).
In this video, we describe a method to identify the small-molecule kinase inhibitors that modulate the apoptosis of self-reactive CD4+CD8+ double-positive immature thymocytes. Apoptosis is induced in the double-positive thymocytes by activating them with anti-CD3- and anti-CD28-coated magnetic beads; this is followed by a small-molecule inhibitor treatment and flow cytometry analysis to detect if the inhibitors modulate the apoptotic marker expression.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Kinase Inhibitor Library Screening (Centrifuge-independent Assay) Treatment of thymocytes with kinase inhibitors Prepare a thymocyte suspension. Dilute the thymocytes in complete RPMI to obtain a thymocyte suspension of 25 x 106 cells/mL. Add 40 μL of th…
Representative Results
Figure 1: Plate layout of the thymocyte activation assay. (Top) Columns 1 and 12 are reserved for controls, while columns 2 to 11 are inhibitor-treated samples (beige). The negative control (nonstimulated [NS]; grey) occupies wells A1 to D1, and the positive control for cell death (dexamethasone-treated [DEX]; purple) occupies wells E1 to H1. Columns 2 to 12 contain thymocytes stimulated with anti-CD3/CD28 beads. The positive co…
Disclosures
The authors have nothing to disclose.
Materials
| RPMI | HyClone | SH30027FS | |
| FBS | HyClone | SH3007103 | |
| L-Glutamine | HyClone | SH3003401 | |
| Sodium pyruvate | HyClone | SH3023901 | |
| 10X PBS | Vivantis | PB0344 – 1L | |
| Kinase Screening Library (96-Well) | Cayman Chemical | 10505 | Exact contents of the library may vary |
| DMSO | Sigma Aldrich | D2650 | |
| Dexamethasone | Sigma Aldrich | D4902 | |
| anti-CD3/CD28 beads | Thermo Fisher Scientific | 11452D | |
| FITC Active Caspase-3 Apoptosis Kit | BD Pharmingen | 550480 | Contains Fixation/Permeabilization buffer, 10X Perm/Wash buffer and anti-caspase 3 antibody |
| DA-Cell Washer | CURIOX | HT1000 | |
| 96-well DA-Cell Plate | CURIOX | 96-DC-CL-05 | |
| Antibodies | |||
| CD3e | BioLegend | 100236 | |
| TCRb | BD Biosciences | 553174 | |
| CD4 | BD Biosciences | 740007 | |
| CD8 | BD Biosciences | 563786 | |
| CD69 | eBioscience | 25-0699-42 | |
| Inhibitors | |||
| TG003 | Cayman Chemical | From the Kinase Screening Library | |
| TG003 | Cayman Chemical | – | From the Kinase Screening Library |
| PKC 412 | Cayman Chemical | – | From the Kinase Screening Library |
| Doramapimod | Cayman Chemical | – | From the Kinase Screening Library |
| Paclitaxel | Cayman Chemical | – | From the Kinase Screening Library |
| Erlotinib | Cayman Chemical | – | From the Kinase Screening Library |
| Necrostatin-5 | Cayman Chemical | – | From the Kinase Screening Library |
| NVP-BEZ235 | Cayman Chemical | – | From the Kinase Screening Library |
| Phthalazinone pyrazole | Cayman Chemical | – | From the Kinase Screening Library |
| AG-879 | Cayman Chemical | – | From the Kinase Screening Library |
| 1-NA-PP1 | Cayman Chemical | – | From the Kinase Screening Library |
| Torin 1 | Cayman Chemical | – | From the Kinase Screening Library |
| Bisindolylmaleimide II | Cayman Chemical | – | From the Kinase Screening Library |
| BIBF 1120 | Cayman Chemical | – | From the Kinase Screening Library |
| SMI-4a | Cayman Chemical | – | From the Kinase Screening Library |
| Bisindolylmaleimide XI (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10657 | Cayman Chemical | – | From the Kinase Screening Library |
| AS-703026 | Cayman Chemical | – | From the Kinase Screening Library |
| Chelerythrine chloride | Cayman Chemical | – | From the Kinase Screening Library |
| Tunicamycin | Cayman Chemical | – | From the Kinase Screening Library |
| GSK 1059615 | Cayman Chemical | – | From the Kinase Screening Library |
| Ruxolitinib | Cayman Chemical | – | From the Kinase Screening Library |
| Necrostatin-1 | Cayman Chemical | – | From the Kinase Screening Library |
| SB 505124 | Cayman Chemical | – | From the Kinase Screening Library |
| INK128 | Cayman Chemical | – | From the Kinase Screening Library |
| Canertinib (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| SB 431542 | Cayman Chemical | – | From the Kinase Screening Library |
| PD 173074 | Cayman Chemical | – | From the Kinase Screening Library |
| Valproic Acid (sodium salt) | Cayman Chemical | – | From the Kinase Screening Library |
| PD 0325901 | Cayman Chemical | – | From the Kinase Screening Library |
| SB 203580 | Cayman Chemical | – | From the Kinase Screening Library |
| VX-702 | Cayman Chemical | – | From the Kinase Screening Library |
| Emodin | Cayman Chemical | – | From the Kinase Screening Library |
| CHIR99021 | Cayman Chemical | – | From the Kinase Screening Library |
| BIO | Cayman Chemical | – | From the Kinase Screening Library |
| Imatinib (mesylate) | Cayman Chemical | – | From the Kinase Screening Library |
| Sunitinib Malate | Cayman Chemical | – | From the Kinase Screening Library |
| Gefitinib | Cayman Chemical | – | From the Kinase Screening Library |
| PP2 | Cayman Chemical | – | From the Kinase Screening Library |
| 3-Methyladenine | Cayman Chemical | – | From the Kinase Screening Library |
| Bisindolylmaleimide I | Cayman Chemical | – | From the Kinase Screening Library |
| Bisindolylmaleimide IV | Cayman Chemical | – | From the Kinase Screening Library |
| Bisindolylmaleimide V | Cayman Chemical | – | From the Kinase Screening Library |
| NSC 663284 | Cayman Chemical | – | From the Kinase Screening Library |
| D 4476 | Cayman Chemical | – | From the Kinase Screening Library |
| NU 7026 | Cayman Chemical | – | From the Kinase Screening Library |
| H-9 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| Indirubin-3'-monoxime | Cayman Chemical | – | From the Kinase Screening Library |
| KN-62 | Cayman Chemical | – | From the Kinase Screening Library |
| KN-93 | Cayman Chemical | – | From the Kinase Screening Library |
| CGP 57380 | Cayman Chemical | – | From the Kinase Screening Library |
| Iso-Olomoucine | Cayman Chemical | – | From the Kinase Screening Library |
| (S)-Glycyl-H-1152 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| Bisindolylmaleimide VIII (acetate) | Cayman Chemical | – | From the Kinase Screening Library |
| ST638 | Cayman Chemical | – | From the Kinase Screening Library |
| SU 6656 | Cayman Chemical | – | From the Kinase Screening Library |
| LY364947 | Cayman Chemical | – | From the Kinase Screening Library |
| SB 203580 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10621 | Cayman Chemical | – | From the Kinase Screening Library |
| YM-201636 | Cayman Chemical | – | From the Kinase Screening Library |
| ZM 447439 | Cayman Chemical | – | From the Kinase Screening Library |
| AS-041164 | Cayman Chemical | – | From the Kinase Screening Library |
| NVP-AEW541 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| PP242 | Cayman Chemical | – | From the Kinase Screening Library |
| ABT-869 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10622 | Cayman Chemical | – | From the Kinase Screening Library |
| 17β-hydroxy Wortmannin | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10626 | Cayman Chemical | – | From the Kinase Screening Library |
| SU 6668 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10572 | Cayman Chemical | – | From the Kinase Screening Library |
| N,N-Dimethylsphingosine | Cayman Chemical | – | From the Kinase Screening Library |
| LY294002 | Cayman Chemical | – | From the Kinase Screening Library |
| U-0126 | Cayman Chemical | – | From the Kinase Screening Library |
| Staurosporine | Cayman Chemical | – | From the Kinase Screening Library |
| KN-92 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| AS-605240 (potassium salt) | Cayman Chemical | – | From the Kinase Screening Library |
| O-1918 | Cayman Chemical | – | From the Kinase Screening Library |
| Y-27632 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| Leelamine | Cayman Chemical | – | From the Kinase Screening Library |
| PD 98059 | Cayman Chemical | – | From the Kinase Screening Library |
| PD 169316 | Cayman Chemical | – | From the Kinase Screening Library |
| TGX-221 | Cayman Chemical | – | From the Kinase Screening Library |
| (S)-H-1152 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| AS-605240 | Cayman Chemical | – | From the Kinase Screening Library |
| D-erythro-Sphingosine C-18 | Cayman Chemical | – | From the Kinase Screening Library |
| OSU03012 | Cayman Chemical | – | From the Kinase Screening Library |
| JNJ-10198409 | Cayman Chemical | – | From the Kinase Screening Library |
| Leelamine (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| Arachidonic Acid Leelamide | Cayman Chemical | – | From the Kinase Screening Library |
| Lauric Acid Leelamide | Cayman Chemical | – | From the Kinase Screening Library |
| AS-252424 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10505 | Cayman Chemical | – | From the Kinase Screening Library |
| PI-103 | Cayman Chemical | – | From the Kinase Screening Library |
| PIK-75 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| Sphingosine Kinase Inhibitor 2 | Cayman Chemical | – | From the Kinase Screening Library |
| Piceatannol | Cayman Chemical | – | From the Kinase Screening Library |
| SC-1 | Cayman Chemical | – | From the Kinase Screening Library |
| (R)-Roscovitine | Cayman Chemical | – | From the Kinase Screening Library |
| BAY-43-9006 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10561 | Cayman Chemical | – | From the Kinase Screening Library |
| AS-604850 | Cayman Chemical | – | From the Kinase Screening Library |
| PI3-Kinase α Inhibitor 2 | Cayman Chemical | – | From the Kinase Screening Library |
| ML-9 | Cayman Chemical | – | From the Kinase Screening Library |
| Triciribine | Cayman Chemical | – | From the Kinase Screening Library |
| Erbstatin Analog | Cayman Chemical | – | From the Kinase Screening Library |
| Kenpaullone | Cayman Chemical | – | From the Kinase Screening Library |
| Olomoucine | Cayman Chemical | – | From the Kinase Screening Library |
| AG-494 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-825 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-1478 | Cayman Chemical | – | From the Kinase Screening Library |
| SB 216763 | Cayman Chemical | – | From the Kinase Screening Library |
| SB 415286 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-17 | Cayman Chemical | – | From the Kinase Screening Library |
| H-8 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| LFM-A13 | Cayman Chemical | – | From the Kinase Screening Library |
| SC-514 | Cayman Chemical | – | From the Kinase Screening Library |
| Apigenin | Cayman Chemical | – | From the Kinase Screening Library |
| AG-18 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10554 | Cayman Chemical | – | From the Kinase Screening Library |
| DRB | Cayman Chemical | – | From the Kinase Screening Library |
| RG-13022 | Cayman Chemical | – | From the Kinase Screening Library |
| RG-14620 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-490 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-82 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-99 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-213 | Cayman Chemical | – | From the Kinase Screening Library |
| AG-183 | Cayman Chemical | – | From the Kinase Screening Library |
| Lavendustin C | Cayman Chemical | – | From the Kinase Screening Library |
| ZM 336372 | Cayman Chemical | – | From the Kinase Screening Library |
| 5-Iodotubercidin | Cayman Chemical | – | From the Kinase Screening Library |
| SB 202190 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10571 | Cayman Chemical | – | From the Kinase Screening Library |
| Nilotinib | Cayman Chemical | – | From the Kinase Screening Library |
| SP 600125 | Cayman Chemical | – | From the Kinase Screening Library |
| L-threo-Sphingosine C-18 | Cayman Chemical | – | From the Kinase Screening Library |
| H-89 | Cayman Chemical | – | From the Kinase Screening Library |
| HA-1077 (hydrochloride) | Cayman Chemical | – | From the Kinase Screening Library |
| AG-370 | Cayman Chemical | – | From the Kinase Screening Library |
| Wortmannin | Cayman Chemical | – | From the Kinase Screening Library |
| AG-1296 | Cayman Chemical | – | From the Kinase Screening Library |
| KT 5823 | Cayman Chemical | – | From the Kinase Screening Library |
| Janex 1 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10574 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10575 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10576 | Cayman Chemical | – | From the Kinase Screening Library |
| NH125 | Cayman Chemical | – | From the Kinase Screening Library |
| TWS119 | Cayman Chemical | – | From the Kinase Screening Library |
| NSC 210902 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10577 | Cayman Chemical | – | From the Kinase Screening Library |
| CAY10578 | Cayman Chemical | – | From the Kinase Screening Library |
| PD 184161 | Cayman Chemical | – | From the Kinase Screening Library |
| CCT018159 | Cayman Chemical | – | From the Kinase Screening Library |
| Myricetin | Cayman Chemical | – | From the Kinase Screening Library |
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Cite This Article
Identifying Kinase Inhibitors that Modulate the Thymocyte Response to Strong TCR Signals. J. Vis. Exp. (Pending Publication), e21605, doi: (2023).