Protocol to detect N-Glycolylneuraminic acid (Neu5Gc) using Flow Cytometry Analysis.

Transcription

1 Page 1 of 5 Protocol to detect N-Glycolylneuraminic acid (Neu5Gc) using Flow Cytometry Analysis. The Gc-Free Basic Pack may be used for immuno-staining cells prior to analysis by Flow Cytometry. This Basic Pack contains essential components that are to be used in concert with other materials, to detect the presence of the non-human sialic acid Neu5Gc on the surface of cells by flow cytometry. The cells of all chordates and some higher invertebrates express sialic acids, a group of acidic sugars on the outermost edge of the cell surface glycocalyx. The two most common naturally occurring forms of sialic acid in mammals are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans cannot synthesize Neu5Gc due to genetic loss of an enzyme required for production from its precursor activated Neu5Ac. Thus, Neu5Gc is a foreign antigen to humans, and all humans tested to date produce variable levels of antibodies against Neu5Gc. However, traces of Neu5Gc enter the human body via dietary sources and Neu5Gc contaminates many bio-therapeutic products and cells. This Chicken anti-neu5gc Primary Antibody provides sensitive and specific identification of Neu5Gc on cell surfaces by flow cytometry. There is no significant cross-reactivity with Neu5Ac. Use of the Blocking Agent in this Basic pack is essential because commonly used endogenous blocking agents invariably contain serum components that can either inhibit detection or introduce contamination from inherent Neu5Gc. Interpretation of positive staining is the visualization of red fluorescence (when Cy5 is used as a readout fluorophore) whereas only background staining should be observable when using the provided isotype Control Antibody on similar cells. This Basic Neu5Gc pack is for Research purposes only, and is not intended for diagnostic use in humans or animals. Materials supplied in the Basic pack: There are 3 Kit Components in the Basic pack. {1 X 15 ml bottles, 2 x 1 ml Eppendorf tubes} To be used for samples in 100 tubes, with 100 microliters per sample with Anti-Neu5Gc and with Control Antibody, diluted as directed in the Blocking Agent. BP-PA: Basic Pack Primary Antibody BP-CA: Basic Pack Control Antibody BP-BA: Basic Pack Blocking Agent Materials required but not included, and Equipment Required for Flow Cytometry: Reagent grade ultra filtered water Disposable 12 x 75-mm cytometry tubes (with filter tops if necessary) Micropipette and tips CHO-K1 cells as positive control, normal human peripheral blood mononuclear cells (PBMCs) as negative controls Secondary Antibody Recommended: Cy5 conjugated anti chicken Jackson Catalog number ( or another secondary based on user preference) Refrigerated Centrifuge at 4 Centigrade. Neu5Gc Flow Cytometry Detection Kit Instructions 1

2 Page 2 of 5 Flow Cytometer and analysis software. PREPARATION FOR IMMUNOSTAINING AND ANALYSIS USING FLOW CYTOMETRY DO NOT USE ANIMAL MATERIALS OR REAGENTS THAT MIGHT CONTAIN Neu5Gc Tissue culture grown CHO-K1 cells can be used as a positive control and human peripheral blood mononuclear cells as the negative control. The tissue culture grown cells grow adherent to the culture flasks or dishes, and are released from the culture flasks or dishes using 5 10 mm EDTA (ethylene-diamine-tetra-acetic-acid), which is allowed to act for 10 minutes at room temperature. Other non-enzymatic methods may be used. Wash cells right away in blocking buffer that contains a lower concentration of EDTA and resuspend cells in blocking buffer to determine cell numbers and viability. Please note: Release of tissue culture cells using trypsin decreases the amount of glycoprotein-bound Neu5Gc. All buffers and reagents used during specimen collection, washing and any other preparation must be free of potential Neu5Gc-containing materials such as fetal bovine serum or bovine serum albumin (BSA). Neu5Gccontaining reagents derived from animal sources will decrease the effective concentration of anti-gc antibodies and lead to erroneously low readings of Neu5Gc levels. Please remember: DO NOT USE Bovine Serum Albumin or Any Other Mammalian products AT ANY STEP. The use of sterile pipette tips and tubes in kit solution preparations can increase the shelf life of reagents and is highly recommended. Keep cap closed on the Blocking Agent while thawing. It is very viscous and must be pipetted slowly for accuracy. Keep the reconstituted solution at 4 degrees centigrade. The blocking solution will be used for the entire staining process. All washes are in Blocking Agent between each reagent incubation step. Each incubation step uses a volume of at least 100 microliters per tube with 1-5 million cells per tube. All washes are performed in a refrigerated centrifuge. All incubations are on ice for at least one hour At the end of the staining procedure, the cells are resuspended in Blocking Agent and taken to the Flow Cytometer for analysis PLEASE FOLLOW THE PROPOSED PROTOCOL AND INSTRUCTIONS as specified in these kits, diligently. Precautions: 1. Do not eat, drink or smoke where samples and kit reagents are handled. 2. Do not use expired or contaminated reagents, or reagents from other kits. 3. Do not pipette by mouth and maintain sterile conditions at all times, to avoid contamination of kit reagents. 4. If handled as recommended, and maintaining sterile conditions at all times to avoid contamination of reagents, the contents of this kit should be stable for up to 6 months after purchase. Neu5Gc Flow Cytometry Detection Kit Instructions 2

3 Page 3 of 5 INSTRUCTIONS FOR REAGENT PREPARATION FROM KIT COMPONENTS A. Wash Buffer (1x PBS, Phosphate Buffered Saline ph ) to be used in all washing steps. B. Diluent Buffer (0.5% Blocking Agent in PBS), by using a cut pipette tip (to allow easy access) and use 0.5 ml of BP-BA: Basic Pack Blocking Agent for every 100 milliliters of Wash Buffer. Prepare prior to use. STORE frozen and can be repeatedly thawed as required C. Primary Antibody (PA): dilute at predetermined concentrations in 100 microliters of Diluent Buffer per tube. STORE reconstituted stock at 4 degrees Centigrade for up to 6 months. D. Control Antibody (CA): dilute at similar concentrations as was predetermined for use for the Primary Antibody, in 100 microliters of Diluent Buffer per tube. STORE reconstituted stock at 4 degrees Centigrade for up to 6 months STAINING PROCEDURE FOR CELLS TO BE ANALYZED USING FLOW CYTOMETRY: IT IS ASSUMED THAT THE USER IS FAMILIAR WITH THE GENERAL PRINCIPLES AND PRACTICE OF FLOW CYTOMETRY Make blocking solution as instructed. Keep at 4 degrees Centigrade. The blocking solution will be used for the entire staining process. Number of cells per tube to be used for immunostaining: 1 5 million cells in one hundred microliters of Diluent buffer per tube. Plans for the immunostaining for flow cytometry analysis should include: A. Tubes containing cells that are the experimental set, to be tested using different dilutions of antibodies, B. One set of tubes containing cells that will be the positive control cells. C. One set of tubes containing cells that will be the negative control cells. Each set of tubes to be analyzed, will then consist of sets of the following : Tube 1 will contain cells that will receive no antibody to be used as unstained control. Tube 2 will contain cells that receive Control Antibody, CA, diluted as instructed. Tube 3 will contain cells that receive Primary Antibody, PA, diluted as instructed. Washing steps: Wash cells by adding 1 ml cold Diluent buffer, and then place in tube holders in a refrigerated centrifuge and centrifuge as recommended for flow cytometry. Carefully remove supernatant and discard. Staining steps: Gently resuspend cells in 100 microliters of Primary Antibody, PA or in 100 microliters of Control Antibody, CA, each of which are diluted in Diluent buffer as determined. Neu5Gc Flow Cytometry Detection Kit Instructions 3

4 Page 4 of 5 Please note: THE DILUTION OF THE PRIMARY ANTIBODY AND THUS THE CONTROL ANTIBODY, WHICH ARE TO BE USED ON DIFFERENT CELL TYPES, CAN RANGE FROM 1:200 TO 1:1600. The investigator must determine the optimum dilution to be used for the cells that are being analyzed. Incubate cells with primary or with control antibodies for at least 1 hour, with the tubes being held on ice. Wash cells as above. SA: Second Antibody (to be purchased: Cy5 conjugated anti-chicken Jackson Catalog number , is recommended for best sensitivity), Gently resuspend cells in 100 microliters of Secondary Antibody diluted in Diluent buffer, and incubate for 1 hour on ice. (The dilution for the secondary antibody is usually the same as for the primary antibody). Wash cells as before. Resuspend cell pellet in 400 ul of Diluent buffer. Read cell fluorescence on a flow cytometer. Analyze using recommended software. NOTES Conditions are described for highly sensitive detection of Neu5Gc on cells with low cell surface levels. For other cell types with high levels (e.g., many mouse myeloma cells), it may be necessary to dilute the primary antibody further. *The end user must determine the concentration and amount of time required to lift adherent cells by EDTA and possibly scraping. Ten minutes with 10 mm EDTA is usually enough to remove cells. The use of a scraper with EDTA is highly advised to avoid excessive cell death. Add 1-5 mm EDTA to blocking solution during preparation and examination of adherent cells. CAUTION: Some cell types will not tolerate higher concentrations of EDTA. STORAGE AND HANDLING The Diluent buffer, Primary Antibody (PA) and Control Antibody (CA) are stored at 2-8 C, away from direct light. Neu5Gc Flow Cytometry Detection Kit Instructions 4

5 Page 5 of 5 The figure is a representative histogram generated using the components in the Basic pack for Flow Cytometry, with the recommended Cy5 labeled second antibody. The Primary Antibody (PA), Control Antibody (CA) and Second Antibody (Cy5 conjugated anti-chicken) were all used at similar concentrations. Negative control cells used here were Human Peripheral Blood Mononuclear Cells (PBMCs) and the results are shown on the top panel. Positive control cells used here were CHO-K1 cells, and Mouse Peripheral Blood Mononuclear cells, and the results are shown on the lower two panels. The Gray shaded area shows the unstained cells which forms the background. The Green solid area represents the positive staining results after immuno-staining with the Anti-Neu5Gc antibody. The Black dashed line shows a negative histogram when similar concentrations of the negative control reagent Chicken IgY were used in Flow Cytometry analysis. Literature cited (The antibody provided is further improved over the one reported in these publications) Martin, M.J., Moutri, A., Gage, F., Varki, A. Human Embryonic Stem Cells Express an Immunogenic Non-Human Sialic Acid Nature Medicine, 11: , Bardor, M., Nguyen, D., Diaz, S., Varki, A. Mechanism of Uptake and Incorporation of the Non-Human Sialic Acid N-glycolylneuraminic acid into Human Cells J.Biol.Chem, 280: , Nguyen, D., Tangvoranuntakul, P., Varki, A., Effects of natural human antibodies against a non-human sialic acid that metabolically incorporates into activated and malignant immune cells. J.Immunol 175: , Neu5Gc Flow Cytometry Detection Kit Instructions 5