Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

This method can help answer key questions in the fields of cell biology and immunology such as how are signaling pathways affected by different stimuli or drugs? The main advantage of this technique is that it allows you to perform single cell signaling analysis in the medium to high throughput fashion. After re-suspending the cells in supplemented RPMI 16/40 medium, transfer the required amount of cell suspension to the wells of a 96 well V-bottom plate.

Transfer the plate to a preheated 37 degree Celsius water bath and allow it to rest there for 10 minutes. Then add 60 microliters of fixed buffer one to each well of a new 96 well plate to set up the fix plate. Place this plate in the 37 degree Celsius water bath.

Transfer a 50 microliter control sample to the plate and mix by pipetting up and down. Optionally, add 10 microliters per milliliter anti-IgM to the cells to start the simulation time course and mix by pipetting up and down. Transfer a 50 microliter sample to the fix plate at each time point, mixing each sample as it is added.

After the last sample is added, leave the fix plate in the water bath for 10 minutes. First, wash the cells three times with PBS. Centrifuge the plate at 500 times G for five minutes and discard the supernatant.

Next, prepare a fresh 96 well V-bottom plate with barcoding reagents, by pipetting five microliters of each barcoding reagent into each well filling the number of wells required to stain each of the samples. In the fixed cell plate, resuspend the cells in each well with 190 microliters of PBS. Then, transfer each well of cells to the corresponding well in the barcoding plate, mixing each well thoroughly.

Let the plate rest at room temperature for 20 minutes in the dark. Centrifuge the plate at 500 times G for five minutes and discard the supernatant. After this, wash the cells in each well twice with the flow wash.

Then add 190 microliters of flow wash to each well of cells. Combine all of the barcoded samples into one 15 milliliter tube and transfer each compensation control to a separate 1.7 milliliter tube. Centrifuge at 500 times G for five minutes and discard the supernatant.

To begin, transfer two milliliters of perm buffer three to a 15 milliliter tube. Store at minus 20 degrees Celsius so it will be ice cold when used. When ready, add 1.5 milliliters of ice cold perm buffer to the 15 milliliter tube containing the barcoded cell population dropwise while vortexing to avoid cell clumping.

Add 100 microliters of ice cold perm buffer to each of the compensation controls in the same manner. Immediately transfer the cells to a freezer at minus 80 degrees Celsius for a minimum of 30 minutes. When ready to begin antigen staining, remove the cells from the freezer and transfer them to a box of ice.

Wash the cells three times with an excess of flow wash. Centrifuge the cells at 500 times G and four degrees Celsius for five minutes. Discard the supernatant and re-suspend the barcoded cell population in a volume of flow wash, such is that there is 25 microliters of cell suspension per phospho-antibody stain.

Re-suspend the compensation controls in 200 microliters of flow wash. To begin preparing antibodies for staining, add 10 microliters of phospho-specific antibody, diluted in flow wash, to each well of a fresh 96 well V-bottom plate. Add 15 microliters of surface marker, diluted in flow wash and 25 microliters of cell suspension to each well for a final volume of 50 microliters per well.

Let the cells rest for 30 minutes at room temperature in the dark. After this, wash the stained cells twice with flow wash. Centrifuge at 500 times G for five minutes.

Discard the supernatant and re-suspend the cells in 150 microliters of flow wash. Then perform flow cytometry analysis as outlined in the text protocol. In the presented protocol, three dimensional barcoding is performed by combining three barcoding dyes.

Individual samples are then de-convoluted by subsequent gating on each barcoding reagent versus side scatter area. The basal and stimulation induced phosphorylation levels of 20 signaling molecules downstream of the B cell receptor are then characterized in B cells from CLL patients and normal controls under various conditions. The stat3 phospho tyrosine 705 is seen to be significantly up regulated.

Cells are stimulated with anti-IgM for up to 30 minutes in order to identify signaling aberrations induced through the B cell receptor pathway. While CLL cells from patients with unmutated IGVH display increased sensitivity towards anti-IgM stimulation, it is only statistically significant for AKT phosphor serine 473. Cells are then exposed to the PI 3-kinase delta inhibitor idelalisib to test if the aberrant AKT pS473 signal can be reversed.

As shown here, the aberrant levels are significantly reduced upon treatment in a concentration dependent manner demonstrating that kinase inhibitors can be applied to normalize aberrant signaling in CLL cells. Before attempting this procedure it's important to remember to titrate barcoding reagents and antibodies, and to verify that selected surface markers are compatible with fixation and permeabilization reagents. The cells need to be in suspension for phosphor analysis, still the protocol can be adapted to aberrant cells or tissue following procedures to obtain single cell suspension, such as whole trypzination.