Alexa fluor 488 labeled goat anti mouse igg h l secondary antibody

Flow cytometry (FCM; FACS Calibur, BD, Franklin Lakes, NJ, USA) was used to determine DDR1 expression in colon cancer cell lines. Cells were resuspended with PBS with 2 mmol·L⁻¹ EDTA. All subsequent steps were carried out at 4 °C. Cells (2 × 10⁵/tube) were incubated with 5 μg·mL⁻¹ primary antibody for 40 min, followed by Alexa Fluor 488‐labeled goat anti‐mouse IgG (H+L) secondary antibody (Zsbio, Beijing, China). All samples were washed three times before being analyzed by FCM. To process the obtained data, novoexpress software (ACEA Biosciences, Hangzhou, China) was utilized.

For determination of in vitro binding ability, cells (2 × 10⁵/tube) were incubated with dilution concentrations ranging from 0.0187 to 40 μg·mL⁻¹ of primary antibodies for 40 min each at 4 °C followed by Alexa Fluor 488‐labeled goat anti‐mouse IgG(H+L) secondary antibody (Zsbio). Mean fluorescence intensity (MFI) of detectable binding of antibodies over multiple test concentrations was measured by FCM. MFI values of samples were subtracted from their respective negative control antibodies and analyzed using the nonlinear regression analysis in prism® software version 5 (Graphpad Software, San Diego, CA, USA).

Briefly, 1×10⁶ HEK293T cells that were transfected with plasmid encoding specified protein were collected using 0.25 mg mL⁻¹ trypsin (Beyotime), followed by two rounds of centrifugation at 3000 rpm for 3 min at 4 °C and washing with 500 µL PBS. Then 1 × 10⁶ cells were incubated with 5 µg mL⁻¹ anti‐S2 primary antibody (1A9) (GeneTex) for 30 min at 4 °C, followed by three rounds of washing with 500 µL PBS to remove unbound primary antibody. Next, cells were incubated with 1.88 µg mL⁻¹ Alexa Fluor 488‐labeled goat anti‐mouse IgG (H+L) secondary antibody (ZSGB‐BIO) or 500 ng mL⁻¹ APC‐labeled goat anti‐mouse IgG secondary antibody (Biolegend) for 30 min at 4 °C. All samples were washed with 500 µL PBS for three times to remove the nonspecific binding antibodies and were monitored by FCM. For determining the expression level of ACE2, 1×10⁶ cells were incubated with 1.07 µg mL⁻¹ rabbit polyclonal anti‐ACE2 primary antibody (SinoBiological), followed by incubating with 1.88 µg mL⁻¹ Alexa Fluor 488‐labeled goat anti‐rabbit IgG (H+L) secondary antibody, the others steps were the same as the aforementioned ones. The NovoExpress software (ACEA Biosciences) was used to analyze the data.