CN111157434A

CN111157434A - Method for detecting luciferase-labeled cells using flow cytometry

Info

Publication number: CN111157434A
Application number: CN202010013013.8A
Authority: CN
Inventors: 王方芳; 黄菁草; 杨艳; 范庆超
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-01-06
Filing date: 2020-01-06
Publication date: 2020-05-15
Anticipated expiration: 2040-01-06
Also published as: CN111157434B

Abstract

The invention discloses a new application of a flow cytometer in detecting luciferase labeled cells, belonging to the field of cell detection. The invention uses the flow cytometer to replace a luciferase labeling instrument to detect the luminescent signal of the reaction of the luciferase in the cells and the substrate, expands the application of the flow cytometer and provides a new method for detecting the luciferase labeled cells.

Description

Method for detecting luciferase-labeled cells using flow cytometry

Technical Field

The invention belongs to the field of cell detection.

Background

Luciferase labelled imaging is a common technique for biomedical research, and generally involves transferring a luciferase (luciferase) gene into cells or tissues, and after expression, if ATP and luciferin are present in the environment of the cells, the reaction emits fluorescence, which can be detected by a light sensitive element.

The transplantation tumor model is to transplant tumor cells into experimental animals to develop tumors (including solid tumors and non-solid tumors), and is often used in various basic oncology studies and test studies of antitumor drugs. The luciferase labeling imaging technology can be used for monitoring the size and the position of the transplanted tumor in the animal body, and the specific method comprises the following steps: tumor cells were labeled with luciferase and tumors with luciferase labeling developed after injection into mice. After a period of time, injecting substrate (fluorescein salt) of luciferase into the abdominal cavity of the mouse or subcutaneously, generating fluorescent product (fluorescein) capable of bioluminescence after the substrate is subjected to the catalysis reaction of luciferase within a few minutes, and photographing by a small animal living body fluorescence imager (IVIS), thereby monitoring the range size and position of the transplanted tumor in the animal body in real time.

Before tumor cells are transplanted into mice, it is important to judge whether luciferase is expressed in the cells and has a catalytic function.

At present, whether cells can successfully express functional luciferase is mainly detected by using a multifunctional enzyme-labeling instrument, but the popularity of the multifunctional enzyme-labeling instrument is low, so that the expression condition of the tumor cell luciferase cannot be estimated in many laboratories, the cells are directly injected into a mouse body for modeling, part of transplanted tumors generated in the later stage cannot be monitored by using fluorescein imaging, and then experimental resource waste is caused.

Disclosure of Invention

The invention aims to provide a method for detecting luciferase labeled cells by using a flow cytometer, which comprises the following specific scheme:

a method of detecting luciferase-tagged cells using flow cytometry, comprising: incubating the cells with the luciferase gene with luciferin salt for 1-10 min, exciting fluorescence by using exciting light with a wavelength of 488nm of a flow cytometer, receiving and emitting fluorescence by using a second channel (529/29nm), and finally processing to obtain a fluorescence signal. [ note: 529/29nm means that the bandpass filter of the channel can receive the emitted fluorescence in the wavelength range of 515nm-544 nm. ]

As mentioned above, the concentration of fluorescein salt is 2.25-3.75 mg/mL.

The method as described above, the cell density is (10-50). times.10⁴one/mL.

As in the previous method, it further comprises the steps of:

1) incubating the cells without the luciferase gene with fluorescein salt for 1-10 min, exciting fluorescence by using exciting light with 488nm wavelength of a flow cytometer, and detecting a fluorescence signal by using a second channel as negative control;

2) cells with luciferase gene were detected directly by flow cytometry using 488nm excitation wavelength and a second channel to detect the fluorescent signal as a blank.

The method comprises the steps of 1) and 2) wherein the cell density is (10-50) x 10⁴Per mL;

and/or the concentration of the fluorescein salt in the step 1) is 2.25-3.75 mg/mL.

The method as described above, wherein the cell is a tumor cell.

Alternatively, the cell is a stem cell.

The method uses the flow cytometer to replace a luciferase labeling instrument to detect the luminescent signal of the reaction of the luciferase in the cells and the substrate, expands the application of the flow cytometer and provides a new method for detecting the luciferase labeled cells.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: and detecting the result by a flow cytometer.

FIG. 2: and (4) in vivo imaging results.

Detailed Description

Example 1 method for detecting luciferase-labeled cells Using flow cytometer

1. Method of producing a composite material

1.1Luciferase labelling of tumor cells

The luciferase gene was constructed into the human acute myeloid leukemia cell line MV4-11 using lentivirus.

The method comprises the following steps:

a. infecting cells with a virus with two reporter genes of luciferase and BSD (blasticidin resistance gene);

b. preparing a culture medium in advance: 1640 basic medium + 10% FBS (fetal bovine serum)

c. MV4-11 cells were plated in one of six wells for a total of 10X 10⁴2mL of the powder; add 100. mu.L of the purchased virus; polybrene was added to the final concentration of 8 ug/mL.

d. Gently mixing, standing at 37 deg.C and 5% CO₂Culturing in a cell culture box, and changing the culture solution after 24 hours.

e.48 hours later, the medium was changed, at which time Blasticidin (Blasticidin) was added to the medium to a final concentration of 10 ug/mL.

f. The screening with Blasticidin was continued for two weeks.

1.2 detection

Collecting (2-10) x 10 with flow tube or EP tube⁴And (3) adding 1 mu L of 15mg/mL substrate (fluorescein salt with the final concentration of 2.25-3.75 mg/mL) into the cells with the volume of 150-250 mu L (the adherent cells need to be digested by pancreatin in advance), standing for 2 minutes, and detecting by using a machine of a flow cytometer (Beckman Co mu Lter, Navios).

The flow cytometer uses a standard 488nm wavelength laser for excitation, and a second channel (529/29nm) for receiving a linear signal of emitted fluorescence. The first test requires a blank and a negative control (blank: cells without substrate; negative control: cells without luciferase with substrate).

1.3 construction and detection of mouse model of transplanted tumor

The positive tumor cells (with fluorescence signals) and the negative control cells obtained by detection are respectively inoculated to the subcutaneous tissues of mice, namely a positive group and a control group, and each group is provided with 5 replicates. After 14 days and 28 days of inoculation, respectively, the mice were injected intraperitoneally with fluorescein salt (200. mu.L, working concentration 15mg/mL) and 5 minutes later, fluorescence was detected on a small animal in vivo fluorescence imager.

2. Results

2.1 flow cytometry results

The flow cytometry results are shown in table 1 and fig. 1. Therefore, over 90% of cells in an experimental sample express luciferase, and the success rate of tumor living body imaging can be improved by using the cells for tumor model construction.

TABLE 1 Positive Rate of Fluoroscein Signal detection by flow cytometry

Note: MFI (mean fluorescence intensity)

2.2 transplantation tumor mouse model detection

After positive cells were inoculated subcutaneously into mice, in vivo imaging was seen (FIG. 2). The flow cytometry detection result is credible, and the effective preliminary screening effect is achieved on the luciferase labeled cells.

It is also inferred from example 1 and general knowledge in the art that the cell type does not affect the effect of the present invention, and it is still feasible to use the method of the present invention for detecting cells other than tumor cells, such as stem cells.

In conclusion, the method uses the flow cytometer to replace a luciferase microplate reader to detect the luminescent signal of the reaction between the luciferase in the cells and the substrate, expands the application of the flow cytometer and provides a new method for detecting the luciferase labeled cells.

Claims

1. a method for detecting luciferase-labeled cells using a flow cytometer, characterized in that:

It includes the following steps: after co-incubating cells with luciferase gene and luciferin salt for 1-10 min, use flow cytometer 488nm wavelength excitation light to excite fluorescence, use the second channel to receive and emit fluorescence, and finally process to obtain fluorescence Signal;

The bandpass filter of the second channel can accept the emission fluorescence in the wavelength range of 515nm-544nm.

2. The method of claim 1, wherein the concentration of the fluorescein salt is 2.25-3.75 mg/mL.

3 . The method of claim 2 , wherein the cell density is (10-50)×10 ⁴ cells/mL. 4 .

4. The method according to any one of claims 1 to 3, wherein:

It also includes the following steps:

1) After co-incubating cells without luciferase gene with luciferin salt for 1-10 min, use flow cytometer excitation light at 488nm wavelength to excite fluorescence, and use the second channel to detect fluorescence signal as a negative control;

2) The cells with the luciferase gene are directly detected by flow cytometer, the excitation light wavelength used is 488 nm, and the second channel is used to detect the fluorescent signal as a blank control.

5. The method of claim 4, wherein:

The cell density in step 1) and step 2) is (10-50)×10 ⁴ cells/mL;

And/or, the concentration of fluorescein salt in step 1) is 2.25-3.75 mg/mL.

6. The method according to any one of claims 1 to 3, wherein the cells are tumor cells.

7. The method according to any one of claims 1 to 3, wherein the cells are stem cells.