Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from "Compendium Immunology Laboratory Guide".
Operation method
Intracellular Fluorescent Stain CFSE for the Detection of Lymphocyte Migration and Reproduction in Lymphocytes Move Basic scheme CFSE labeling of lymphocytes Material (see Appendix 1 for items with V) Laboratory animals, human peripheral blood, or cultured lymphocytes. VPBS, pH 7.4 H a n k s Equilibrium Salt Solution (H B S S ) p H 7. 4 PBS with 5 % (V/V) heat-inactivated FBS 5 m m o l / L C F S E storage solution 0.5 m o l / L P B S solution of E D T A (for cultured lymphocytes) Antigens and mitogens for experiments Oml plastic tubes (optional) Fluorescence microscope with fluorescence filters Note: Cells labeled with C F S E cannot be detected immediately because the fluorescence intensity is very high at that time. Most of the C F S E taken up by the cells is not yet stable and will be lost gradually in the following days. la. For large cell numbers: Prepare lymphocytes as described in Appendix 2H, Module 2.1 and Module 8.1, suspending human PBMC with PBS (serum free) to 50 X 106 cells/m l and mouse lymphocytes with HBSS (serum free) to the same concentration. lb. For low cell counts: Suspend freshly isolated lymphocytes in PBS containing 5 % FBS (the protein attenuates the toxicity of C F S E) to a concentration of 0-5 X 106 ~ IOXlO6 cells/ml. lc. Cultured lymphocytes: do not centrifuge resting lymphocytes. Add the labeling reagent directly to the medium containing 1 0 % F B S . 2 . Dilute the 5 m m o l /L C F S E stock solution with P B S at a ratio of 1 / 1 0 0 (50 Mmol/L concentration). Add 110ul of the above solution per ml of cell suspension and mix rapidly. For example, add the cell suspension to an IOml plastic tube, keeping the tube upright, and add C F S E to the dry part of the tube wall. Keep the tube upright and cover it with a cap, then quickly invert it several times to mix. Alternatively, add an equal volume of C F S E pre-diluted to lOumol/L while shaking the cells. 3a Separated lymphocytes: incubate at room temperature for 5 min, add 10 times the volume of PBS containing 5% FBS, centrifuge at 300 g for 5 min at 20℃, and discard the supernatant. Wash three times, each time add 10 times the volume of 5% FBS PBS, 20 ℃, 30 g centrifugation for 5 min, discard the supernatant. 3b. Cultured lymphocytes: wash with PBS and incubate with 0.5 mmol/L EDTA in PBS solution for 5 min to remove clumps. Wash with PBS again and suspend in culture medium. 4 . C F S E labeled lymphocytes are suspended in protein-free (serum-free) tissue culture medium and injected intravenously (i.v .) into the recipient animal. ) into the recipient animal. If the subject is a mouse, I X l O 6 to 40 X 106 cells are injected i.v. into the recipient animal at 0.1 to 0.5 mm. 1 to Two different populations of lymphocytes can be tracked simultaneously by using different concentrations of C F S E labeling. One population of cells is labeled with 5 u m o l / L of C F S E (Steps I a and I b), and the other population is labeled with either 1/4 (1.25;xmol/L) or 1/16 (0.312jumol/L) of the usual agricultural concentration. To avoid fluorescence intensities outside the detection range on the flow cytometer when detecting cells relayed to animals within 24 h, label with 1/4 (1.25umol/L) or 1/8 (0.625 Umol/L) of the commonly used concentration (Steps 1 and 2). 5 . Observe the location of C F S E-labeled cells in lymphoid organs and other tissues by fluorescence microscopy with filters or by immunohistochemical examination with specific antibodies to incendin (Garton and Schoenwolf, 1996; Graziano, 1998). To visualize CFSE-labeled cells by fluorescence microscopy, animal organs are removed and cut into approximately 3-mm slices, which are placed on slides for observation. Fluorescence microscopy results in rapid extinction of C FSE fluorescence, which is completely extinguished by conventional histologic staining. H 33342 (Parish, 1999) is recommended for short-term localization analysis at low resolution. For higher resolution localization studies, immunohistochemical examination of CFE-labeled cells on histological sections is recommended. 6 . Suspend CFE-labeled cells in culture medium and stimulate them with antigen or mitogen in vitro (Modules 2.7, 2.11, 8.8, 8.9)0 . 7a. Obtaining cells in vivo: Collect lymphoid organs and prepare single cell suspensions (Appendix 2H and Module 2.1). 7b. Harvesting the same in vitro cultured cells: collect cultured cells, wash with 3 ml of PBS, suspend with 2 ml of 0.5 mmol/LE DTA/PBS, incubate at 37°C for 5 m i n to disaggregate the cell clusters. Incubate at 37°C for 5 m i n to disaggregate the cell mass. Centrifuge at 300 g for 5 min at 20°C, suspend the cells with PBS containing 5 %FBS and transfer to a flow tube. If manual counting with a hemocyte counter plate is required (Appendix 3A), a small amount of the cell suspension should be placed in another centrifuge tube or V-bottom plate and kept on ice. 7c. Harvesting Multiple In Vitro Cultured Cells: To collect cells from a 96-well flat-bottom plate, aspirate an appropriate amount of supernatant and transfer the cells from three wells to one well of a 96-well V-bottom plate. Wash again with 150 M1 EDTA as in Step 7b. Centrifuge the plate at 300 g for 2 min. Pipette the supernatant along the wall of the well from the top down until the tip of the gun reaches the junction of the vertical wall and the V-bottom. Vibrate the plate on an oscillator to mix the cells, followed by addition of the next washing reagent using a multichannel pipette. This protocol summarizes the experimental steps for quantitative analysis of the proliferation of C F S E labeled cells. See Chapter 4 for more information on flow cytometry analysis. C F S E labeled cells (see basic program) Flow cytometric analyzer capable of detecting trichromatic fluorescence Flow Cell Sorter (optional) 1 . Perform flow labeling of cells according to experimental needs (e.g., cell surface labeling, Table 4.1; intracellular cytokine staining, Table 5.8; propidium iodide staining for apoptosis, Table 2.14). 1; intracellular cytokine staining, Unit 5.8; propidium iodide staining for apoptosis, Unit 2.14). Perform flow cell analysis (Module 4.2). For software analysis 2a. Analyze C F S E peaks with appropriate software (e.g., Quantumsoft's Profit, SPSS Science's Peakfit, Verity SoftwareHouse's ModFit, Science Speak's C F S E Modeler) to analyze the C F S E peaks. For manual calculations 2b. record the geometric mean of the fluorescence intensity of C F S E labeled control cells, as well as of the fluorescence intensity of unlabeled control and stimulated cells (autofluorescence of unlabeled proliferating cells is higher than that of unlabeled control nonproliferating cells). 3b. Subtract the corresponding unlabeled cell control (e.g., 600-2 = 598) from the geometric mean of the fluorescence intensity of the C F S E-labeled control cells. Convert the resulting value to the logarithm of 10 (in this case, 2.776). 4b. Determine the geometric mean fluorescence intensity of the daughter cells. The fluorescence intensity of the daughter cells is 1/2, 1/4 ...... and so on of the peak fluorescence of the nonproliferating cells, so that the fluorescence intensity is subtracted by 0.3logl0 per division. For more product details, please visit Aladdin Scientific website.
If the above method is to be used for large numbers of cells, the lymphocyte concentration should be increased from 50 X 106 cells/m l to 1,000 XIO6 cells/m l . If C F S E-labeled cells are scheduled to be detected within 24 h, they should be labeled with 1/4 or 1/8 of the commonly used C F S E concentration.
0.2 m l of fluid into the tail vein (injection of more than 50 X 106 cells saturates the mouse lymphoid organs; the number of cells injected before this saturation point is linearly related to the number of cells entering the lymphoid organs).
The limitation of the cell frequency is that it can only be analyzed before the fluorescence intensity of dividing cells overlaps with the spontaneous incandescence of unstimulated cells.