Anti-Neu5Gc Antibody Kit Protocol: Flow Cytometry

 

Reagent List

Anti-Neu5Gc Antibody Kit (Cat. No.146901) FITC Goat anti-chicken IgY Antibody (Cat. No. 410802), or other fluorescently labeled anti-chicken IgY secondary antibody PBS Positive and negative control cells (see Notes)

 

Notes

Anti-Neu5Gc may be used for staining cells prior to analysis by flow cytometry. This kit contains the components needed to identify Neu5Gc on the surface of cells by flow cytometry.

Use of the blocking agent (Neu5Gc Assay Blocking Solution) provided in the kit is essential, as commonly used blocking agents invariably contain serum, or serum components, that can either inhibit detection or introduce Neu5Gc contamination.

Tissue culture-grown CHO-K1 cells can be used as a positive control, and human peripheral blood mononuclear cells serve as a negative control. Adherent tissue culture-grown cells should be released from the culture flasks by using 5-10 mM EDTA for 10 minutes at room temperature. Immediately wash cells in blocking buffer that contains a lower concentration of EDTA and resuspend cells in blocking buffer to determine cell numbers and viability.

It is assumed that the user is familiar with the general principles and practices of flow cytometry.

 

Protocol Steps

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1. Prepare a 1X solution of the Neu5Gc Assay Blocking Solution in PBS.
   (Warm the 40X blocking solution to room temperature prior to use)
   For example, add 10 µL of Neu5Gc Assay Blocking Solution (40X) to 390 µL of PBS.


2. The following tubes should be set up for flow cytometry analysis:
   • Three tubes containing cells to be examined.
   • Three tubes containing positive control cells.
   • Three tubes containing negative control cells.
   Note: If multiple dilutions of the primary antibody are tested, prepare more tubes.


3. Wash cells in cold PBS and adjust each cell suspension to a concentration to 1 x 10⁷ cells/mL in PBS.


4. Transfer 100 µL of cells to appropriately labeled tubes.


5. Add 10 µL of the 1X Neu5Gc Assay Blocking Solution made in step one to each tube and incubate for 10 minutes at room temperature. Proceed directly to the next step. Do not wash out the blocking solution.


6. Gently resuspend cells in 50 µL of appropriate diluted antibody as outlined below:
   • Tube 1 - Unstained control (optional, but highly recommended); add 50 µL of PBS.
   • Tube 2 - Isotype control antibody.
   • Tube 3 - Anti-Neu5c antibody.
   For example, to stain in a 1:400 dilution, add 3.2 µL of antibody to 400 µL of PBS and add 50 µL of the diluted antibody to each tube containing 110 µL of cells/PBS plus blocking solution.
   
   Note: The optimal dilution of the antibody can range from 1:200 to 1:1,000. Investigators must determine the optimum dilution for cells being analyzed.
   
   Tip: If this is the first time running this experiment, optimize by using different dilutions of primary antibody. More tubes containing sample will be needed.


7. Incubate cells on ice for at least 1 hour.


8. Wash cells as above.


9. Gently resuspend cells in 100 µL of Secondary Antibody in PBS, and incubate for 1 hour on ice.
   
   Note: The dilution of the antibody can range from 1:200 to 1:1,000. Investigators must determine optimum dilution for cells being analyzed.


10. Wash cells as before.


11. Resuspend cell pellet in 400 µL of PBS.


12. Analyze cells on a flow cytometer.

Note: If excessive background staining is observed, increase the amount of blocking buffer used. In step 3, wash and resuspend cells in 1X Neu5Gc Assay Blocking Solution and incubate for 10 minutes at room temperature. Dilute antibodies in 1X Neu5Gc Assay Blocking Solution and add to cells in step 6, skipping step 5. Replace PBS with blocking solution in subsequent wash/resuspension steps.