Lectin and Galectin information

Staining Procedure

# We use FITC-Lectin for staining, so please avoid using green fluorescence cells for GBP FACS analysis.

Cell number requirements:

1x10e6 for one lectin staining

I. Cell Processing

1. Wash adherent cells with Trypsin and then add Trypsin to incubate at 37 °C for 2-3 minutes (2 ml for 10 cm dish, 1 ml for 6-well dish).

2. Transfer to a centrifuge tube filled with 2% BSA in PBS to a total volume of 10 ml.

3. Centrifuge at 350G for 5 minutes (Suspension cells start from this step).

4. Remove the supernatant.

5. Mix 1 ml of 2% BSA in PBS to suspend the cells uniformly.

6. Count the cells and adjust the cell concentration to [1x10e7 cells/ml].

II. Preparation of Staining Reagent and Staining

1. Adjust the stock concentration to [specific concentration] using 2% BSA in PBS.

2. Quickly add 100 μl of the dye into 100 μl of cell suspension and vortex immediately.

3. Place on ice in the dark for 10 minutes.

4. Add 500 μl of 2% BSA in PBS.

5. Centrifuge at 400G, 4°C for 5 minutes.

6. Remove the supernatant.

7. Repeat steps 4 to 6 twice, with centrifugation speeds of 450G and 500G respectively.

8. Add 500 μl of 2% BSA in PBS to homogenize before analysis.

Data Output and analysis

HeLa cell treated with Mannosidase I inhibior (kifunensine) for one and five days, followed by PFA fixation the glycan profile on cell surface were examined by lectins and galectin staining.

References and examples