3.2 Production of a Cleared Cell Lysate
Peter Simons, Virginie Bondu, Angela Wandinger-Ness, Tione Buranda
Rho GTPase
Rab GTPase
Cell signaling
Cytoskeleton
Hantavirus
Flow cytometry
Integrin activation
Sepsis
Multiplex
Protease-activated receptors
PARs
Thrombin
Argatroban
Bead functionalization
Glutathione-S-transferase
GST
GTPase effector beads
Rap1
RhoA
Rac1
Rab7
Fluorescence calibration beads
Abstract
Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione- S -transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.
Section 3.2 'Production of a Cleared Cell Lysate' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee
Steps
3.2 Production of a Cleared Cell Lysate
Two days before an assay seed a 48-well plate with 20,000 target cells in 100 μL of culture medium per well, resulting in about 50,000 cells the next day. The rate of cell proliferation might vary based on cell type and conditions. The critical target is 50,000 cells at the start of an experiment.
Remove the culture medium and replace with 100µL . Specific inhibitors of signaling can be added to the cells for the desired amount of time depending on the requirements of the reader’s assay to establish proper inhibition before stimulation.
After stimulating the cells ( see Section 3.5 in Protocol "Applications of G-Trap Assay" for examples), chill the plate in an ice/water bath. Add 100µL to each well with a 1 mL pipette and triturate the mixture gently to achieve homogenous lysis of the cells.
Transfer the lysate into a 0.65 mL microfuge tube and centrifuge at 5000x g. The 200 μL of cleared lysate is enough for triplicate assays using 50 μL for each test.
