Lectin binding was evaluated by flow cytometry as described previously48 (link). The following reagents were used at the final concentrations indicated: allophycocyanin-streptavidin (APC-Strep) (Life Sciences, S-868), 5 ug/mL), LEA-biotin (Vector labs B-1175, 1 μg/mL), L-PHA rhodamine (Vector labs RL-1112, 20 μg/mL). Empty vector (EV) control and LKB1 WT expressing H460 and H2122 cells, and doxycycline-inducible GFPT2 KO H460 and H157 cells were harvested by centrifugation, washed twice with DPBS, then resuspended in DPBS at 2.0 × 106 cells/ml. Then, 200 μL of cell suspension was transferred to a V-bottom 96-well plate and pelleted by centrifugation at 600g for 5 minutes. The cell pellets were incubated with 100 μL of lectin diluted in DPBS for 60 min at 4 °C (LEA incubation was 30 min), then washed with DPBS three times. When a secondary detection reagent was used, cells were incubated with 5 μg/mL APC-Strep in DPBS for 45 min at 4 °C. Fluorescence was analyzed by flow cytometry on a FACS Aria II SORP with dual lasers at 488 nm and 635 nm. Plots show the mean fluorescence intensity (MFI), typically for 10,000 cells.
L pha rhodamine
Manufactured by Vector Laboratories
L-PHA rhodamine is a fluorescent label commonly used in cell biology and molecular biology applications. It binds specifically to L-PHA (Phaseolus vulgaris leukoagglutinin), a lectin that recognizes glycoproteins with β-1,6-branched N-linked oligosaccharides. The rhodamine dye attached to L-PHA provides a means to detect and visualize the distribution of these glycoproteins in cells and tissues.
Automatically generated - may contain errors
3 protocols using l pha rhodamine
1
Lectin Binding Evaluation by Flow Cytometry
2
Lectin Binding Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lectin binding was evaluated by flow cytometry. The following reagents were used at the final concentrations indicated: SNA-biotin (Vector Laboratories B-1305, 20 μg/ml), MAL-II-biotin (Vector Laboratories B-1265, 10 μg/ml), allophycocyanin-streptavidin (APC-Strep) (Life Sciences, S-868), 5 μg/ml), LEA-biotin (Vector Laboratories B-1175, 1 μg/ml), L-PHA rhodamine (Vector Laboratories RL-1112, 20 μg/ml). K20 cells were cultured for the times indicated, with or without supplemental sugar, as indicated. Cells were harvested by centrifugation, washed twice with DPBS, then resuspended in DPBS at 2.0 × 106 cells/ml. Then, 200 μl of cell suspension was transferred to V-bottom 96-well plate and pelleted by centrifugation. The cell pellets were incubated with 100 μl of lectin diluted in DPBS for 60 min at 4 °C (LEA incubation was 30 min) then washed with DPBS 3 times. When a secondary detection reagent was used, cells were incubated with 5 μg/ml allophycocyanin-streptavidin (Life Sciences, S-868) in DPBS for 45 min. Fluorescence was analyzed by flow cytometry on a FACSCalibur instrument (BD Biosciences) equipped with dual lasers at 488 nm and 635 nm. For sialidase experiments, before lectin staining, cells were incubated with Sialidase A (Prozyme GK80040) for 90 min at 37 °C in DPBS containing 0.1% BSA. Plots show the mean fluorescence intensity (MFI), typically for 10,000 cells.
3
Lectin Binding Evaluation by Flow Cytometry
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!