Flow cytometry (FCM) is an advanced instrument developed in the 1980s by high-tech technologies such as monoclonal antibodies, fluorescent chemistry, lasers, and computers. It has been widely used in immunology, biochemistry, biology, oncology, and blood. Research and clinical routine work. The detection of human leukocyte surface markers can quickly and correctly diagnose leukemia and lymphoma, accurately classify lymphocyte populations and subpopulations, and isolate and purify a certain group or subgroup of cells. Live cell immunofluorescence technology is used for specimen preparation for FCM detection. It can also be observed under fluorescence microscope after staining. Under certain experimental conditions, the specificity and sensitivity of live cell immunofluorescence staining are better than droplet fixation. Conventional indirect immunofluorescence result.

(a) Principle

The surface of living cells retains a relatively complete antigen or receptor, and first binds to the corresponding antigen on the cell surface with a specific murine monoclonal antibody, and then binds with the fluorescently labeled second antibody, according to the measured fluorescence intensity and positive percentage. The density and distribution of the corresponding antigens are known.

(2) Operation steps

Preparation of highly active cell suspensions (cultured cell lines, peripheral blood mononuclear cells,

Thymocytes, spleen cells, etc. can be used in this method)

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Adjust the cell concentration with 10% FCS RPMI1640

5×106～1×107/ml

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Add 40 μl of cell suspension to pre-specific McAb (5-50 μl)

Small glass tube or plastic centrifuge tube, add 50μl 1:20 (with DPBS)

Dilute) inactivate normal rabbit serum

↓4°C 30min

Wash twice with washing solution, add about 2ml of washing solution each time

1000rpm × 5min

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Discard the supernatant and add 50 μl of working concentration of goat anti-mouse

(or rabbit anti-mouse) fluorescent marker, fully shaken

↓ 4°C 30min

Wash twice with washing solution, add about 2ml each time

1000rpm × 5min

↓

Add appropriate amount of fixative (if the specimen is prepared for FCM, generally added

1ml fixative, such as observation under a fluorescence microscope after tableting,

Add 100-500μl fixative to the cell concentration)

↓

FCM detection or observation under fluorescence microscope after filming

(The specimen can be stored in a test tube for 5 to 7 days)

(3) Reagents and equipment

1. Various specific monoclonal antibodies.

2. Fluorescently labeled goat anti-mouse or rabbit anti-mouse secondary antibody, inactivate normal rabbit serum.

3. 10% FCS RPMI1640, DPBS, washing solution, fixative (see appendix).

4. Glass tubes, plastic tubes, centrifuges, fluorescence microscopes, etc.

(four) matters needing attention

1. The whole operation was carried out at 4 ° C, and NaN₃ was added 10 times higher than the conventional preservative dose in the washing solution. The above experimental conditions were to prevent cross-linking and shedding of the primary antibody after binding to the cell membrane antigen.

2. Wash enough to avoid the combination of free antibody blocking secondary antibody and primary antibody on the cell membrane, and false negatives.

3. Add appropriate amount of normal rabbit serum to block certain cell surface immunoglobulin Fc receptors, reduce and prevent non-specific staining.

4. Cell activity is better, otherwise non-specific fluorescent staining is prone to occur.

Attached:

1. DPBS (×10, stock solution)

NaCl 80g

KCl 2g distilled water added to 1000ml

Na₂HPO₄ 11.5g diluted 1:10 with distilled water

KH₂PO₄ 2g

2. Washing liquid

DPBS 900ml

FCS 50ml (final concentration 5%)

4% NaN₃   50ml (final concentration 0.2%)

3. Fixing solution

DPBS 1000ml

Glucose 20g (final concentration 2%)

Formaldehyde 10ml

NaN₃ 0.2g (final concentration