Research

Immunological examination of MSC

Experiment 1. CFSE labeling to detect T lymphocyte proliferation experiment

1. Experimental principle

Fluorescent dye CFSE (CFDA-SE) is a new type of dye that can fluorescently label living cells. CFSE can easily penetrate the cell membrane, covalently bind to intracellular proteins in living cells, and release green fluorescence after hydrolysis. In the process of cell division and proliferation, its fluorescence intensity will gradually decrease with the division of cells, and the labeled fluorescence can be evenly distributed to the two daughter cells, so its fluorescence intensity is half of that of the parent cell. It can be used to detect cell proliferation, cell cycle estimation and cell division.

2. Reagent consumables

Fresh sterile human blood 10ml; Celltrace CFSE cell proliferat 1 kit (Invitrogen, Cat. No. C34554); Lymphocyte separation medium Ficoll-Paque PLUS (GE, Cat. No. 17-1440-03); RPMI-1640 medium (Gibco, Cat. No. C11875500BT) ); Fetal Bovine Serum (Gibco, Cat. No. 10100147C); Dynabeads® Human T-Activator CD3/CD28 (Gibco, Cat. No. 11131D); Penicillin-Streptomycin-Glutamine (100X) Liquid (Gibco, Cat. No. 10378016); Sodium Pyruvate (100 mM) ) (Gibco, Cat. No. 11360070); HEPES (1M) (Gibco, Cat. No. 1894147); 2-Mercaptoethanol (55 mM) (Gibco, Cat. No. 1799301); MEM Non-Essential Amino Acids (100X) (Gibco, Cat. No. 11140050); none Bacterial PBS (Solebold, Item No. P1020-500);

Complete-medium configuration: RPMI-1640, with 10% FBS, also contains

Penicillin-Streptomycin-Glutamine (100X) 1:100

Sodium Pyruvate (100 mM) 1:100

HEPES (1M) 1:100

2-Mercaptoethanol (55 mM) 1:1000

MEM Non-Essential Amino Acids (100X) 1:100

CFSE working solution: 50 μl of DMSO was added to 1 vital CFSE power to be a 5mM stock solution. The final concentration used in this experiment was 20uM, that is, diluted 250 times.

Apparatus: CO2 incubator (Thermo Fisher Scientific, model 3111), centrifuge (Thermo Fisher Scientific, model Heraeus multifuge X1R), flow cytometer (BD, model FACSAriIII)

3. Experimental method

(1) Anticoagulation with EDTA in a vacuum blood collection tube, and collect 8ml of fresh blood (two tubes, 4ml/tube). Add 4ml of sterile PBS to each tube to dilute, spread on 6ml of lymphocyte separation medium, centrifuge at 400g for 20min, increase to 2 and decrease to 0.

(2) Gently aspirate the middle layer of the separation solution and the lymphocytes into a 15ml sterile centrifuge tube, fill with PBS, and centrifuge at 1500rpm for 10min. Resuspend cells in PBS and wash again.

(3) Lymphocytes were resuspended with Complete-medium and counted (usually 0.5~1 million cells can be obtained from 1ml of blood).

(4) CFSE staining: add 4 μl CFSE working solution to 96 μl Complete-medium and mix evenly; adjust the obtained lymphocytes to 900 μl, add CFSE staining solution, mix quickly, and place at room temperature for 5 minutes. Then, Complete-medium was added to wash the cells for 3 times, and some cells were taken to observe the cell staining by flow cytometry.

(5) Lymphocyte stimulation culture: The lymphocytes stained with CFSE were adjusted to a concentration of 1×106 cells/ml with Complete-medium. Use magnetic beads to activate T cells (Human T-Activator CD3/CD28): follow the instructions to explore the pre-experimental conditions, explore the appropriate concentration of beads, and add 2 μl beads to stimulate 105 cells. According to experience, when lymphocytes are cultured for 3 days, it is appropriate that 3-4 CFSE splitting peaks will appear in the cell flow detection.

(6) Mixing ratio of MSCs and lymphocytes: According to literature reports, three concentration ratios of MSCs:lymphocytes: 1:2, 1:4, and 1:6 are planned to be used in the experiment. MSCs were pre-passaged in 48-well plates and cultured overnight. Lymphocytes stained with CFSE and evenly mixed with beads were gently added to the culture plate plated with MSC cells according to the proportion of cell numbers, and finally the medium ratio of MSCs and T cells was 1:1, and cultured for 3 days.

(7) After the incubation, collect the lymphocytes in the plate. The pipette should avoid touching the bottom of the well plate and avoid collecting MSCs that grow adherently to the wall. Pipette the medium several times in the up and down directions to collect the medium, centrifuge at 4°C and 1500 rpm for 5 min, and collect the lymphocytes. The supernatant was collected for Elisa to detect the content of TNF-α in the medium. The collected cells were divided into two, one cell was used for flow cytometry detection of lymphocyte proliferation, and the other cell was used for cell cycle analysis.

4.Experimental Results

Experiment 2. Inhibition test of lymphocyte secretion of TNF-a

1. Experimental principle

Elisa method was used to detect the changes of TNF-a in the culture medium after lymphocytes were co-cultured with MSCs. Tumor necrosis factor alpha (TNF-a) antibody was coated in a 96-well microplate to make a solid phase carrier, and standard substances or samples were added to the microwells respectively, and the TNF-a and TNF-a were connected to the solid phase carrier. After the unbound biotinylated antibody was washed, HRP-labeled avidin was added, and TMB substrate was added to develop color after thorough washing again. TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The shade of color was positively correlated with the TNF-a in the sample. Measure the absorbance (O.D. value) with a microplate reader at a wavelength of 450 nm, and calculate the sample concentration.

2. Reagent consumables

Human TNF-a ELISA kit (Wuhan Yuncloning, Cat. No. SEA133Hu), multi-function microplate detector (TECAN).

3. Experimental method

The cell culture medium supernatant collected in experiment 1 was used as a sample for the experiment. The specific operation was carried out according to the instructions of the kit, and a standard curve was prepared, and three replicate wells were made for each sample to be tested. Absorbance was measured at 450 nm, and inhibition was calculated.

4.Experimental Results

Experiment 3. Lymphocyte cycle test

1. Experimental principle

Propidium iodide (PI) can bind to intracellular DNA and RNA. After RNA is digested by RNase, the fluorescence intensity of PI bound to DNA detected by flow cytometry directly reflects the amount of intracellular DNA. Due to the difference in DNA content in each phase of the cell cycle, normal cells in G1/G0 phase usually have the DNA content of diploid cells (2N), while G2/M phase has the DNA content of tetraploid cells (4N), while S Phase DNA content is between diploid and tetraploid. Therefore, when the intracellular DNA content is detected by flow cytometry PI staining, each phase of the cell cycle can be divided into G1/G0 phase, S phase and G2/M phase, and each phase can be calculated by software percentage.

2. Reagent consumables

Propidium iodide PI solution (1mg/ml) (Solebold, catalog number C0080-1ml), flow cytometer (BD, model FACSAriIII)

3. Experimental method

A portion of lymphocytes collected in experiment 1 was washed once with PBS and centrifuged at 4°C at 1500 rpm for 5 min. Dry the PBS as much as possible and fix the cells with 70% ethanol for 1 h at 4°C or overnight at -20°C. Then wash the cells twice with PBS, add 200 μl of PBS containing 100 μg/mL RNase I to each tube of cells, incubate in a water bath at 37°C for 1 h, add 1 ml of PBS to wash the cells, and then add 200 μl of PI staining solution (final concentration 10 μg/mL) at room temperature Incubate in the dark for 15 min, add 1 ml of PBS to wash the cells once, resuspend the cells with 500 μl of PBS, and then detect by flow cytometry. The obtained data were analyzed with ModFit software.

4.Experimental Results

Experiment 4. Karyotype analysis of mesenchymal stem cells

1. Experimental principle

Colchicine inhibits mitosis, disrupts the spindle, and arrests chromosomes in metaphase. In such mitosis, although the chromosomes are longitudinally split, the cells do not divide and cannot form two daughter cells, so the chromosomes are doubled, which can be used for the analysis of chromosome karyotypes in metaphase.

2. Reagent consumables

Colchicine (Aladdin, Cat. No. C106739), Potassium Chloride (Sinopharm, Analytical Grade), Carnoy's Fixative (Solebo, Cat. No. G2312), Giemsa Stain (Solebo, Cat. No. G1015); Inverted Fluorescence Microscope (Nikon, Ti-S)

3. Experimental method

(1) Plate the MSC cells to be tested and culture overnight to obtain a cell monolayer (the cell density is about 70%), add colchicine (final concentration 20ug/mL), continue to culture for 10-12h, and collect the cells.

(2) Swollen cells: Resuspend cells in sterile 0.3% KCl solution (gently resuspend), incubate at 37°C for 30 minutes, and collect cells by low-speed centrifugation at 800-1000 rpm for 10 minutes.

(3) Add 2 mL of pre-chilled Carnoy fixative to the cells, and gently resuspend the cell pellet (do not pipette). Cells were collected by centrifugation at 800-1000 rpm for 10 min at low speed for 20 min at 4°C.

(4) Add 1 mL of pre-chilled Carnoy fixative, and gently resuspend the cell pellet (do not pipette). Drop 2 drops of the above cell suspension onto a pre-cooled glass slide at a height of 10-15 cm, immediately blow and shake to disperse the cells, and dry at room temperature.

(5) Giemsa staining: configure 1× Giemsa staining solution, add the staining drop to the slide, stain for 40-50 min, rinse with running water for 1-2 min after staining, and dry at room temperature.

(6) Take pictures and observe and count: put the prepared slides under a microscope for observation, and count the chromosome metaphases (at least 100), count the number of chromosomes, and count the proportions.

(7) Take pictures and print the results, cut them out and paste them in pairs to make a karyotype. Comparison of the karyotype of normal cells to draw conclusions.

4. Experimental results