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…

Divulgazioni

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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Citazione di questo articolo
Identifying Kinase Inhibitors that Modulate the Thymocyte Response to Strong TCR Signals. J. Vis. Exp. (Pending Publication), e21605, doi: (2023).

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