Live cell immunofluorescence technique-flow cytometry specimen preparation

Summary

Flow cytometry (FCM) can be used for: (1) basic research and clinical application of immunology, especially in combination with monoclonal antibody technology; (2) in immunophenotyping, sorting, and immune monitoring of tumor cells; (3) monitoring of the immune status of the body; and (4) phylogeny and characterization of immune cells.

Operation method

Live cell immunofluorescence technique-flow cytometry specimen preparation

Principle

The surface of living cells retains a more complete antigen or receptor, first use specific mouse-derived monoclonal antibody to bind to the corresponding antigen on the surface of the cell, and then use fluorescently labeled secondary antibody to bind to it, and according to the measured fluorescence intensity and the percentage of positivity, the density and distribution of the corresponding antigen can be known.

Materials and Instruments

Cell Samples
Rabbit serum Secondary antibody Monoclonal antibody FCS-RPMI1640 DPBS
Glass tubes Plastic tubes Centrifuge Fluorescence microscope

Move

Preparation of highly active cell suspensions (cultured cell lines, peripheral blood mononuclear cells, thymocytes, splenocytes, etc. can be used in this method)

1. Adjust the cell concentration to ^{5×106-1×107/ml} with 10% FCSRPMll640.

2. Take 40 μl of cell suspension and add it to a small glass tube or plastic centrifuge tube with specific McAb (5-50 μl), then add 50 μl of 1:20 (diluted with DPBS) inactivated rabbit serum.

3. Allow to stand at 4℃ for 30 min.

4. Wash twice with washing solution, each time add 2 ml of washing solution, 1000 r/min×5 min. 5.

5. Discard the supernatant, add 50 μl of sheep anti-mouse (or rabbit anti-mouse) fluorescent marker at working concentration, and shake well.

6. Leave at 4℃ for 30 min.

7. Wash with washing solution twice, each time adding 2 ml of liquid, 1000 r/min × 5 min. 8.

8. Add appropriate amount of fixative (if preparing specimens for FCM, generally add 1 ml of fixative; if observing under fluorescence microscope after preparation, add 100-500 μl of fixative depending on the concentration of cells).

9. FCM test or fluorescence microscope observation after preparation, (specimen can be stored in the test tube for 5--7 days)

Caveat

1. The whole operation is carried out at 4℃, and the washing solution is added with NaN3 which is 10 times higher than the amount of conventional preservative. The above experimental conditions are to prevent cross-linking and detachment of the primary antibody after binding the antigen on the cell membrane.

2. The washing should be sufficient to avoid the combination of the free antibody-enclosed secondary antibody and the primary antibody on the cell membrane, which may result in false negative.

3. Adding appropriate amount of normal rabbit serum can close the immunoglobulin Fc receptor on the surface of certain cells, reduce and prevent non-specific staining.

4. The cell activity should be good, otherwise non-specific fluorescence staining is easy to occur.

Common Problems

Source Laboratory Animals and Animal Experimental Methodology

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