Methods:
Materials
The following were purchased: PIPES, bovine serum albumin (BSA), EGTA, EDTA,
D(+)glucose, and fMLP (formyl-met-leu-phe) (Sigma-Aldrich, St. Louis, MO);
crystallized human serum albumin (HSA) (Miles Laboratories, Elkhart, IN); antiCD203c (clone NP4D6, BioLegend, San Diego, CA); mouse IgM anti-human IgE,
HP6061P (Hybridoma Reagent Laboratory, Baltimore, MD); anti-CD123-PE (mIgG1
isotype) (BD Biosciences, Sparks, MD), CRA-1, anti-FceRIa (eBiosciences, San Diego,
CA).
Buffers & Reaction Conditions
PIPES-albumin-glucose (PAG) buffer consisted of 25 mM PIPES ((piperazine-N,Nbis-2-ethanesulfonic acid), 110 mM NaCl, 5 mM KCl, 0.1% glucose, and 0.003% HSA.
PAGCM was PAG supplemented with 1 mM CaCl2 and 1 mM MgCl2. PAG-EDTA
(ethylenediamine N, N, N’, N’- tetraacetic acid) consisted of PAG supplemented with
4 mM EDTA, elutration buffer, PAG containing 0.25% BSA. PBS (phosphate buffered
saline) was 20 mM phosphate buffered isotonic saline (pH 7.4) and was often
supplemented with 4% bovine serum albumin (Sigma, St. Louis, MO). Reactions
were performed in either PAGCM buffer (final volumes of 0.1 ml) or when longer
incubations were needed, in RPMI-1640 media supplemented to contain 1 mM Ca++
(final volume of 0.1 ml).
Flow cytometry
Each sample of the stored suspension of fixed cells was first labeled with a mixture of
1/100 dilution of CRA-1 (mIgG2b anti-FceRI Ab) and 1/40 dilution of anti-CD123-PE
(IgG2a isotype) for 15 minutes. After a single wash, the cells were divided equally for
resuspension in PBS-0.04% BSA or permeabilization buffer (part B of the the
Fix&Perm kit from Caltag Laboratories, now Life Technologies, Grand Island, NY).
For each of these conditions, the cells were incubated with either isotype control
IgG1Ab (biotin-labeled) or anti-CD203c-biotin (1/500 dilution from manufacturer’s
stock solution) in PBS-BSA buffer or permeabilization buffer. After a 60 minute
incubation, the cells were washed and resuspended in PBS-0.04% BSA and incubated
overnight at 4˚C with secondary reagents, anti-mIgG2b-alexa488 and streptavidinalexa647. Analysis by flow cytometry used FS/SS, CD123 (red) and FceRI (green)
positivity to gate for basophils, as described previously (figure E1). Note that pilot
studies showed that this anti-CD203c antibody detects CD203c equally well on fixed
and non-fixed cells.
Statistics
In general, data is shown as the mean ± standard error of the mean, both in text and
figures. Ward hierarchical clustering was performed on the fractional results
(external CD203c/total CD203c) for all data points and the final cluster (fractions
greater than 0.30) to be joined to entire group was excluded from the line fitting
routine.
Figure E1: Basophil gating strategy: rightmost histogram shows both the FL4
(alexa647 distributions) for both IgG2b control antibody (very light grey) and
CD203c antibody (darker grey).
Figure E2: Change in cell surface and total CD203c expression during stimulation.
Panel A: results for stimulation with 1 µM FMLP and sampled after 5 minutes of
stimulation (n=7). Panel B: results for stimulation with 1 µg/ml anti-IgE Ab and
sampled after 10 minutes of stimulation (n=3). There is a change in the total cellular
content of CD203c after stimulation and the difference between the total CD203c
before and after stimulation is labeled as lost CD203c and is shown as a fraction of
the initial total in the light gray bars for each panel.