Evaluation of lymphocyte-mediated cytotoxicity using flow cytometry
Evaluation of lymphocyte-mediated cytotoxicity using flow cytometry
Cytotoxicity of lymphocytes, is the killing of target cells by the release of particles containing perforin and granzyme and/or through the interaction of Fas-Fas ligands. Currently, methods for assessing lymphocyte-mediated cytotoxicity use flow cytometry.
Principle
The basic principle of using flow cytometry to assess lymphocyte-mediated cytotoxicity is the use of fluorescence-derived caspase substrates that permeabilize cells and multiparametric flow cytometry to detect caspase activation in target cells and to quantify and display cytotoxic lymphocyte activity by these means.
Operation method
Evaluation of lymphocyte-mediated cytotoxicity using flow cytometry
Principle
The basic principle of using flow cytometry to assess lymphocyte-mediated cytotoxicity is the use of fluorescence-derived caspase substrates that permeabilize cells and multiparametric flow cytometry to detect caspase activation in target cells and to quantify and display cytotoxic lymphocyte activity by these means.
Materials and Instruments
Equipment: FACSCalibur flow cytometer. Move The experimental steps for assessing lymphocyte-mediated cytotoxicity using flow cytometry were as follows: A. Resuspend the target cells in RPMI 1640 complete culture solution T 15 mL pointed bottom tubes. B. Count the live cells by Tapan blue rejection staining method. Adjust the cell concentration to 2 x 106/mL. C. Add 0.5 mL of target cells to the FACS tube as tube A. Place tube A on ice until the top of the ice. Place tube A on ice until sampling on the flow cytometer. D. Add 0.5 mL of target cells to a 15 mL sharp-bottomed tube as tube B. Add an apoptosis inducer such as a chemotherapeutic agent. Add apoptosis inducer such as staurosporine to induce apoptosis at a final concentration of 1uM. Incubate at 37°C with 5% CO2 for 3-5 h. (Tube B serves as a control for flow setup of FL1 channel). E. Divide the remaining target cell suspension into two portions into 15 mL pointed-bottom tubes (tubes a and b). Add CTO/TFL2 at a final concentration of 1.5uM to the tubes. Add the antigen peptide at the final concentration in tube a and the irrelevant control peptide in tube b. F. Incubate tubes a and b at 37°C with 5% CO2 for l h after loosening the caps, and prepare effector cells during this time. G. After incubation of target cells for l h, wash at least once with 10 times volume of complete RPMI 1640. H. Resuspend cells with complete RPMI 1640 at 2 x 106/mL. place cells on ice until effector cells are ready. I. Remove 0.5 ml of cells from each of tubes a and b into FACS tubes (C and D). C is placed on ice until sample (this tube is used to set up the flow-through FL-2 channel control), and D is placed on ice (this tube is used to assay the basal amount of target cell apoptosis).
Reagents: Erythrocyte lysate, complete culture medium, Tepan blue.
A. Collect spleens and lymph nodes 5-8d after viral infection.
B. Place 1 70uM cell strainer on a 50 mL tube and place freshly removed organs into the cell strainer.
C. Grind tissue with a 3 mL syringe bolus until mostly fibrous tissue remains.
D. Rinse the cells on the screen with 20 mL of complete RPMI 1,640 culture solution and discard the screen.
E. Centrifuge at 250 g for l0 min, discard supernatant.
F. Resuspend splenocytes in RBC lysate (5 mL/spleen to remove erythrocytes), incubate at room temperature for 5 min, top up with RPMI 1,640, centrifuge at 250 g for l0 min, and discard supernatant.
G. Resuspend the cells with appropriate amount of RPMI 1 640 and count the viable cells by placenta blue reject method.
H. Adjust the cell concentration to 5x107/mL.
(iii) Detection of Apoptosis in Target Cells with Caspase SubstrateA. Add a series of dilutions of effector cells to a 96-well round bottom plate according to the E/T ratio. Use a multichannel sampler to transfer 100 cells/well from row A to the corresponding wells in row B. Mix 3-5 times. Mix 3-5 times. Change the pipette tip and transfer 100 ul of cell suspension from row B to row C at 100 ul/well.
B. Add 100ul/well of target cell suspension (2 x 105/well) to a 96-well plate. Blow up and mix target and effector cells.
C. Add 200ul of target cells from tube D to one well of the 96-well plate.
D. Incubate the plate at 37°C for 1-3 h with 5% CO2.
E. Centrifuge the plate and tube B at 250 g. Gently shake the plate and vacuum aspirate the tube to remove the supernatant.
F. Add 75ul of caspase substrate PhiPhiLux-GlD2 to each sample and mix gently. Do not oscillate the mixed samples from this step to avoid apoptotic cell fragmentation.
G. Incubate at 37°C with 5% CO2 for 30 min.
H. Wash the cells twice with ice pre-cooled buffer for each sample.
I. After washing, resuspend the cells with wash buffer and add 300ul per sample.
J. Transfer the cells into FACS tubes. Label the astaxanthin-treated target cells as B tube.
(iv) Flow cytometry analysisA. Use tube A (unlabeled target cells) to set the initial values of FL-1 and FL-2 so that the fluorescence intensity of the majority of events is located at 100-101 for each axis.
B. Use tube B (PhiPhiLux-G1D2 labeled apoptotic target cells) to set compensation for FL-2 channels.
C. Use tube C (CT0/TFI2 labeled target cells) to set compensation for FL-1 channels.
D. Collect the remaining samples.
(E) Detection of cytotoxic activityA. Analyze the data with CellQuest, FlowJo or WinMDI software to obtain quadrant statistics for different cell populations.
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