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.
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)
2. Stimulation of thymocytes using anti-CD3/CD28 beads
3. Setup of the plate washer
NOTE: The instructions for setting up the plate washer are provided by the manufacturer. The steps are mentioned in brief below. Roughly 150 mL of solution is needed for each priming step.
4. Staining of surface antigens
5. Fixation of cells
6. Intracellular staining for active caspase 3
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 control for thymocyte activation (stimulated samples [α-CD3/CD28]; green) occupies wells A12 to D12, and the vehicle control (stimulated and DMSO-treated [α-CD3/CD28 + DMSO]; red) occupies wells E12 to H12. (Bottom) Flow cytometry plots of active caspase-3 (ActCasp3), CD69, and TCRβ staining of thymocytes gated within the double-positive (DP) gate. Representative plots of the different controls are shown. NS = nonstimulated; DEX = dexamethasone-treated samples; α-CD3/CD28 + DMSO = samples stimulated with CD3/CD28-coated beads and treated with DMSO; α-CD3/CD28 = samples stimulated with CD3/CD28-coated beads.
Figure 2: Thymocyte viability after treatment with inhibitors. (A) Experimental outline of the major steps in the screening assay. There are three proposed methods for the stimulation and staining of the thymocytes used in the activation assay, namely (1) the culturing of thymocytes in standard 96-well plates, followed by staining using a conventional centrifugation-based protocol, (2) the culturing of thymocytes in standard 96-well plates, followed by staining using a centrifugation-independent washing protocol, and (3) the culturing of thymocytes in small-volume plates, followed by staining in the same plates using a centrifugation-independent washing protocol. (B) Gating strategies used in the viability assays. The live cell gate was derived from the forward scatter (FSC) and side scatter (SSC) plots. Inhibitors that were deemed to be too toxic at the tested concentration were subject to further viability assays at 10-fold lower concentrations. Representative inhibitor-treated samples are shown. Note the common control (DMSO-treated [DMSO]) used for the 1 µM and 0.1 µM samples. (C) Plate layout of diluted inhibitors. A schematic representation of the plates of inhibitors diluted in DMSO to a concentration of 500x the intended final concentration. Each well represents one unique inhibitor; the grey wells are empty. The concentrations shown are the final concentration when added to the cell cultures, namely 10 µM (dark red), 1 µM (fuchsia), and 0.1 µM (blue).
The authors have nothing to disclose.
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 |