Goat anti human igg γ chain specific pe conjugated secondary antibody

Example 4

In this example, CHO-CXCR3 cells were lifted from culture flasks using non-enzymatic Cell Dissociation Buffer—PBS based (Life Technologies #13151-014). Cells were resuspended in FACS Buffer (2% Fetal Bovine Serum in PBS) with sodium azide at 1×10⁶ cells/ml and 800 μl were aliquoted into a tube for each antibody. Antibody was added to a final concentration of 5 μg/ml and incubated at room temperature for 1 h. The samples were placed on ice and a 105 μl aliquot at various time points was added to 190 μl FACS buffer in a 96-well plate and centrifuged for 3 min. Supernatant was discarded, the cells were resuspended in 105 μl of FACS buffer, duplicate aliquots (50 μl) added to another 96-well plate containing 150 μl FACS buffer and incubated at room temperature. After all of the time points were collected, the plate was centrifuged for 3 min, supernatant discarded and the cells resuspended in 50 μl goat anti-human IgG (γ-chain specific)-PE conjugated secondary antibody (Southern Biotech #2040-09) diluted 1:500 in FACS Buffer. Cells were incubated for 30 min at 4° C. and then washed 1× with FACS Buffer. The cells were resuspended in a final volume of 30 μl FACS Buffer and analyzed using the Flow Cytometer. The binding of clone 32B12 over time is shown in FIG. 1.

Example 3

In this example, CHO-CXCR5 cells were lifted from culture flasks using non-enzymatic Cell Dissociation Buffer—PBS based (Life Technologies #13151-014). Cells were resuspended in FACS Buffer (2% Fetal Bovine Serum in PBS) with sodium azide at 1×10⁶ cells/ml and 800 μl were aliquoted into a tube for each antibody. Antibody was added to a final concentration of 5 μg/ml and incubated at room temperature for 1 h. The samples were then placed on ice and a 105 aliquot at various time points was removed and added to 190 μl FACS buffer in a 96-well plate and centrifuged for 3 min. Supernatant was discarded, the cells were resuspended in 105 μl of FACS buffer, aliquoted (50 μl) in duplicates into another 96-well plate containing 150 μl FACS buffer and incubated at room temperature. After all of the time points were collected, the plate was centrifuged for 3 min, supernatant discarded and the cells were resuspended in 50 μl goat anti-human IgG (γ-chain specific)-PE conjugated secondary antibody (Southern Biotech #2040-09) diluted 1:500 in FACS Buffer. Cells were further incubated for 30 min at 4° C. and then washed 1× with FACS Buffer. The cells were resuspended in a final volume of 30 μl FACS Buffer and analyzed using the Intellicyt Flow Cytometer. The binding of various IgG1 clones over time is shown in FIG. 1.

Example 14

In this example, CHO-CCR2 cells were lifted from culture flasks using non-enzymatic Cell Dissociation Buffer—PBS based (Life Technologies #13151-014). Cells were resuspended in FACS Buffer (2% Fetal Bovine Serum in PBS) with sodium azide at 1×10⁶ cells/ml and 800 μl were aliquoted into a tube for each antibody. Antibody was added to a final concentration of 5 μg/ml and incubated at room temperature for 1 h. The samples were then placed on ice and a 105 μl aliquot at various time points was added to 190 μl FACS buffer in a 96-well plate and centrifuged for 3 min. Supernatant was discarded, the cells were resuspended in 105 μl of FACS buffer, aliquoted (50 μl) in duplicates into another 96-well plate containing 150 μl FACS buffer and incubated at room temperature. After all of the time points were collected, the plate was centrifuged for 3 min, supernatant discarded and the cells were resuspended in 50 μl goat anti-human IgG (γ-chain specific)-PE conjugated secondary antibody (Southern Biotech #2040-09) diluted 1:500 in FACS Buffer. Cells were further incubated for 30 min at 4° C. and then washed 1× with FACS Buffer. The cells were resuspended in a final volume of 30 μl FACS Buffer and analyzed using the Intellicyt Flow Cytometer. The off-rate of various IgG1 clones are shown in FIG. 13.