Envision reader

Cells were cultured at a density of 5 × 10³ cells per well in flat-bottomed 96-well plates in DMEM supplemented with 10% FBS with antimycotic antibiotic. The experiment was done at different time points (24 h and 48 h or based on cell doubling time). CellTiter 96® Aq_ueous One Solution Reagent (Promega, Madison, WI) was added and the experiment was conducted according to the manufacturer’s instructions.
CellTiter-Glo assay (Promega Madison USA) for determining cell proliferation was conducted according to the manufacturer’s instructions. Briefly, CellTiter-Glo reagent was added directly to the wells of 96 well plate and luminescence was measured on an Envision reader (PerkinElmer).

To generate target cells expressing luciferase, lentiviral luciferase was transduced into NALM-6. In 96-well plates, CAR NK-92, CAR KHYG-1, or CAR T cells were co-cultured with luciferase-expressing NALM-6 at indicated E:T ratio and incubation time. At the end of incubation time, the Bright-Glo Luciferase assay reagent (E2650; Promega) was added to each well containing effector/target cell mixture. After shaking the plate for 5 min at room temperature, luminescence was detected using an EnVision reader (PerkinElmer).

The TR-FRET-based
LANCE Ultra assay (Perkin Elmer) was used to determine
IC₅₀ values for various CSF1R inhibitors. Kinase activity
and inhibition in this assay were measured as recommended by the manufacturer.
Briefly, a specific Ultra ULight GT peptide substrate (50 nM final
concentration) was allowed to get phosphorylated by CSF1R (0.5 nM
final concentration) in enzymatic buffer (50 mM HEPES pH 7.5, 10 mM
MgCl₂, 1 mM EGTA, 0.01% Tween 20, 2 mM dithiothreitol,
1% DMSO) containing ATP at the concentration of the K_m value (25 μM) of the kinase for 1 h at room temperature.
All compounds were tested in an 8-point dose–response curve
up to a final concentration of 10 μM. The compound transfer
was facilitated via acoustic dispensing with the Echo 520 (Beckman
Labcyte) using the Echo Dose–Response software package. Subsequently,
phosphorylation or inhibition was detected by the addition of specific
europium (Eu)-labeled anti-phospho antibodies (2 nM), which upon binding
to the phospho-peptide gives rise to a FRET signal. The FRET signal
was recorded in a time-resolved manner in a Perkin Elmer EnVision
reader. All assays were performed in a final volume of 20 μL
in low-volume white 384-well plates from Corning (4513). All assay
data were analyzed with the Quattro Workflow software package from
Quattro Research.

The binding of multiple co-regulator motif peptides to FXR-LBD protein in response to compounds was determined by AlphaScreen assay (the schematic diagram is shown in Figure 1B) using a hexahistidine detection kit (Perkin-Elmer, code 6760619R). For screening (natural product library from BioBioPha company, China. http://www.biobiopha.com/ (accessed on 14 November December 2022)), small molecules were transferred into a 384-well OptiPlate by Echo520 (Beckman Coulter, Inc. California, USA), the mixture containing FXR-LBD and biotin-SRC2-3 was transferred into the OptiPlate wells by a multidrop microplate dispenser (Thermo Fisher, USA), and AlphaScreen signaling was measured using an Envision reader (Perkin-Elmer, USA) after incubation for 1 h at room temperature. The assay was performed using approximately 20~40 nM 6His-FXR-LBD protein and approximately 20 nM biotinylated co-regulator motif peptides in the presence of 2~5 μg/mL donor beads (streptavidin-coated) and acceptor beads (nickel-coated) in a buffer containing 50 mM MOPS, 50 mM NaF, 0.05 mM CHAPS, and approximately 0.1 mg/mL BSA, pH 7.4. The following biotinylated co-regulator motif peptides were used:
SRC1-2, biotin-SPSSHSSLTERHKILHRLLQEGSP
SRC2-3, biotin-QEPVSPKKKENALLRYLLDKDDTKD
NCoR-2, biotin-GHSFADPASNLGLEDIIRKALMGSF

TR-FRET assays were performed as described previously^{11 (link)}. In brief, samples were transferred to a low volume 384 well plate (Greiner) in serial dilutions starting from a defined concentration (1–4 µg/µl for total lysates and homogenates and 2 ng/µl for recombinant proteins) and the equivalent of one fifth of sample volume of antibody cocktail was then added. Soluble HTT was measured with 2B7-Tb/MW1-D2 and 2B7-Tb/4C9-D2 using 0.17 ng/µl of 2B7-Tb and 1.7 ng/µl of D2 labelled antibody. TR-FRET measurements were routinely performed following 1 hour incubation at 20 °C or overnight at 4 °C using an EnVision Reader (Perkin Elmer). Data analysis and evaluation of the temperature- and polyQ-dependent variation in TR-FRET signal were performed as previously described^{11 (link)}. The points in the curves correspond to the averages of the background-subtracted fluorescence ratio (665/620) relative to the samples replicates and the bars represent the standard deviation among these replicates. The bar graphs represent the temperature- and polyQ-dependent variation and the standard deviations among values obtained in different experiments.