Method for amplifying PBMC by virtue of CD40 in combination with PD-L and cell factors
A cytokine, PD-L technology, applied in animal cells, vertebrate cells, cell culture active agents, etc., can solve the problem of high ratio of negative co-stimulatory molecules PD-1/PD-L1, achieve low equipment requirements, The effect of high collection rate, simple operation and time saving
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0037] Example 1: In vitro expansion and culture of adoptive immunotherapy cells
[0038] Isolation, extraction and expansion of PBMC
[0039] Peripheral blood from normal people was taken, diluted with PBS at a ratio of 1:2, and then subjected to Ficoll density gradient centrifugation. First add Ficoll, which has been placed at room temperature in advance, into the Fahrenheit tube, and then slowly add the diluted blood along the tube wall according to the ratio of Ficoll:diluted blood = 1:2 (the Ficoll tube is inclined at 45°, and the interface between Ficoll and the diluted blood should not be strongly shaking), and then centrifuged in a temperature-controlled centrifuge (1800rpm / min, 30min, 20-24°C). After centrifugation, 4 layers of liquid are obtained as shown in the figure below. The PBMC we need comes from the middle cloud layer. Carefully absorb the cloud layer cells with a pipette, then wash and centrifuge with 10 times the volume of PBS, and finally obtain PBMCs. ...
Embodiment 2
[0052] Example 2: Detection of immunophenotype of adoptive immunotherapy cells by flow cytometry
[0053] Flow cytometric detection of molecules on the membrane surface of cells
[0054] ① Transfer the cell suspension to a centrifuge tube, centrifuge at 1500rpm / min for 3min, and discard the supernatant.
[0055] ②Add PBS (containing 1% calf serum), centrifuge at 1500rpm / min for 3min, remove the supernatant, and repeat this step once more.
[0056] ③Set control group: negative control tube (only add unstained cells), single-label compensation tube.
[0057] ④Add PBS (containing 1% calf serum) to make a final concentration of 1×10 6 cells / ml of cell suspension.
[0058] ⑤Take 100μl of the cell suspension in step 4 and add it to a new tube.
[0059] ⑥ Add fluorescent antibody to the cell suspension and incubate for 30 min at room temperature in the dark.
[0060] ⑦Wash and centrifuge, add 500μl sheath fluid, and test on the machine.
[0061] On the 14th day of culture, the ...
Embodiment 3
[0075] Example 3: Detection of killing function in vitro
[0076] Flow cytometric detection of expression of IFN-γ in T lymphocytes
[0077] ① Add 100 μl of the prepared cell suspension to each flow loading tube, the number of cells is about 1x106.
[0078] ② Label surface antigens (CD3, CD4, CD8) according to the cell surface antigen staining method, and the dosage of fluorescent antibody is 2 μl / test.
[0079] ③ Wash the cells with pre-cooled PBS.
[0080] ④ Vortex to resuspend the cells, add 200 μl of Fixation / Permeabilization working solution (Fixation / Permeabilization), and mix again.
[0081] ⑤ Incubate at 4°C for 12 minutes in the dark.
[0082] ⑥ Add 500 μl Permeabilization Buffer working solution, centrifuge to wash the cells and discard the supernatant.
[0083] ⑦Repeat step 6 to wash the cells.
[0084] ⑧Add 100 μl of diluted fluorescent-labeled IFN-γ antibody (diluted with Permeabilization Buffer working solution) directly, and incubate at 4°C for 20 minutes i...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More