Donor BALB/c, third party SJL and congenic B6.Kd thymocytes
were isolated and 1x106 cell aliquots were incubated with
100µl of serially diluted recipient serum or with non-diluted cell
culture supernatant. Fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse IgG, phycoerythrin (PE)-conjugated anti-mouse IgG1, FITC-conjugated
anti-mouse IgG2a, FITC-conjugated anti-mouse IgG3 and biotinylated anti-mouse
IgG2b followed by PE-Streptavidin conjugate were used as detecting antibodies at
a 1:50 – 1:100 dilution (all from BD Pharmingen). Rat anti-mouse IgG2a
monoclonal Ab R19-15 used in our studies was raised against pooled BALB/c and
C57BL/6 Ig and thus has reactivity to both IgG2a and IgG2c. The staining was
performed as previously published by our group (46 (link), 47 (link)). Cells were washed,
fixed in 1% paraformaldehyde, and analyzed by flow cytometry. For every
sample and every IgG isotype, the mean fluorescence intensity (MFI) of each
dilution was determined. The dilution that returned the MCF to the level
observed when thymocytes were stained with an 1:90 dilution of naïve B6
serum was divided by two and reported as the titer. Binding to B6.Kdtarget cells was presented as mean fluorescence intensity (MFI) at different
serum dilutions.
were isolated and 1x106 cell aliquots were incubated with
100µl of serially diluted recipient serum or with non-diluted cell
culture supernatant. Fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse IgG, phycoerythrin (PE)-conjugated anti-mouse IgG1, FITC-conjugated
anti-mouse IgG2a, FITC-conjugated anti-mouse IgG3 and biotinylated anti-mouse
IgG2b followed by PE-Streptavidin conjugate were used as detecting antibodies at
a 1:50 – 1:100 dilution (all from BD Pharmingen). Rat anti-mouse IgG2a
monoclonal Ab R19-15 used in our studies was raised against pooled BALB/c and
C57BL/6 Ig and thus has reactivity to both IgG2a and IgG2c. The staining was
performed as previously published by our group (46 (link), 47 (link)). Cells were washed,
fixed in 1% paraformaldehyde, and analyzed by flow cytometry. For every
sample and every IgG isotype, the mean fluorescence intensity (MFI) of each
dilution was determined. The dilution that returned the MCF to the level
observed when thymocytes were stained with an 1:90 dilution of naïve B6
serum was divided by two and reported as the titer. Binding to B6.Kdtarget cells was presented as mean fluorescence intensity (MFI) at different
serum dilutions.