We used stimulation with anti-IgE or IL-3 in whole blood, which would allow for the potential interaction of multiple cell types under conditions that simulate an allergic response. 

One milliliter aliquots of whole blood were distributed into round-bottom tubes with loose lids and warmed in a 37°C water bath for 30 seconds. Freshly prepared, 1 mL aliquots of pre-warmed RPMI 1640 (Gibco) were supplemented with polyclonal rabbit anti-IgE (Bethyl Laboratories) or IL-3 (PeproTech). The prewarmed 1 mL of whole blood was then mixed with either 1 mL of RPMI 1640, or RPMI 1640 supplemented with anti-IgE (final concentration, 1 μg/mL) or IL-3 (final concentration, 2 ng/mL) and incubated for 30 minutes at 37°C in a 5% CO2 incubator. After the incubation period, red blood cells were removed by hypotonic lysis with 20 mL of cold RBC lysis buffer (BioLegend) and incubation for 15 minutes on ice. The lysis reaction was stopped with PBS supplemented with EDTA (Invitrogen Life Technologies; final concentration, 2 mM) followed by centrifugation at 250g for 5 minutes at 4°C (all centrifuge runs on live cells were done with these conditions).

*Blood was stored at most 24h at 4 deg C

*Anticoagulant EDTA or heparin can have distinct impact on basophil activation profiles

*We chose to use whole blood vs. cells post red blood cell lysis because we wanted to retain as close of an environment as that in situ

*Must use sterile techniques

*If followed by mass cytometry analysis vs flow cytometry, watch out for metal contamination. Must use low barium PBS, avoid glass bottles 

Stimulation of basophils + a bunch of other cells in vitro

DNA stains enable a commonly used FACS method of monitoring a compound’s effects on cell cycle progression. 


1. Transfer cells from culture dishes to FACS tubes. Make sure you pipet vigorously to get all the lightly adherent cells. The protocol assume 0.5 million cells per sample, so scale up or down with the volumes for your samples. 

2. If needed, stain for surface antigens.

3. Spin cells for 5 minutes, 500 g in a microcentrifuge. Discard supernatants afterwards.

4. Resuspend the cell pellet in 250 uL wash buffer (1x PBS with 5mM EDTA). 

5. Add 125uL 100% COLD ethanol and vortex immediately. Repeat once. 

6. Incubate at room temperature for at least 30 minutes. This cell suspension can be left at 4 C overnight instead if necessary.

7. Spin cells at 5 min, 500 g. Discard supernatants afterwards.

8. Add 250 uL propidium iodide solution (0.1 mg/ml propidium iodide in wash buffer). Place samples on ice.

9. The exact parameters and protocol at this point are dependent on your flow cytometer, but in general start with gating out cell debris using SSC/FSC and doublets using FL2-H/FL2-W. PI has an excitation wavelength of 488nm and maximal emission at 617nm.

10. Collect at around 10,000 events at low speed settings using cell count vs FL2-H as the primary indicator of cell cycle. There will be 2 primary peaks representing G0/G1 and G2 phase respectively, with the G2 phase having double the FL2 height due to having twice the amount of DNA. Cells with less FL2 height than the G1 population are sub-G1 (dying) and cells in between the two main peaks are in the S phase.

Cell Cycle Progression Analysis

A commonly used quantifiable metric for protein phosphorylation is achieved through the use of fluorescence tagged antibodies specific for phosphorylated protein sites. Post treatment cell samples incubated with these antibodies can be analyzed via flow cytometry to measure their respective levels of phosphorylation. Be sure to include unstained controls and secondary antibody only controls if necessary.


1. Treat 0.5-2x106 cells per condition for 2-3 hours.

2. Collect cells to conical tubes / eppitubes and spin down cells (500g, 5 minutes).

3. Prechill 810µl Methanol in -20 C freezer.

4. Resuspend pellet in 100µl PBS (does not have to be sterile).

5. Add 100µl of 4% paraformaldehyde (PFA)  -or-  25µl of 16% PFA to each sample.

6. Incubate 10 min at room temperature.

7. Add 800µl of ice cold methanol 100% methanol. At this stage, you can store at -20oC indefinitely or incubate samples for 30 minutes before proceeding to the next step.

8. Spin down cells 12,000rpm, 5min. This spin should be as fast as possible to ensure maximum sample collection.

9. Add 1 ml PBS to each sample.

10. Spin down cells 12,000rpm, 5min.

11. Prepare 100 uL of primary antibody master mix per sample. Concentrations may need to be 2-5 times higher than an equivalent western blot procedure.

12. Discard sample supernatant and add 100µl of diluted antibody in PBS. 

13. Incubate 30 minutes in the dark on ice (or a fridge).

14. Wash by adding 1 mL PBS and incubate for 5 minutes at room temperature. Spin down cells at 8000 rpm, 5 minutes. Prepare secondary antibody solution in the meantime if necessary (100 uL solution per sample).

15. Remove supernatant. If using secondary antibody repeat resuspension, incubation, and wash steps with secondary antibody solution. If proceeding to FACS analysis, resuspend in 250 uL PBS.

16. Analyze via FACS.

Phosphoflow Protocol

The scientist with profound knowledge gained from a multinational and multidisciplinary career in top research institutions with expertise in innate immunity and immunology, virology, cancer, and skin biology. Leading a group for the development of the three-dimensional skin, cancer, and immunology platforms. Established novel approaches in drug discovery in skin care-related products. Experience in scientific writing, project development, and management.  PI on NIH SBIR grant.

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