Detection and enrichment of hematopoietic stem cells by marginal group cell phenotyping
Detection and enrichment of hematopoietic stem cells by marginal group cell phenotyping
Detection and enrichment of hematopoietic stem cells by marginal group cell phenotyping is based on the isolation of hematopoietic stem cells from mouse bone marrow based on the characterization that an important dye, Hoechst 33342, can be pumped by stem cells. Currently, flow cytometry is used for the detection and enrichment of hematopoietic stem cells by marginal population cell phenotyping.
Principle
The basic principle of Hematopoietic Stem Cell Detection and Enrichment by Marginal Population Cell Phenotyping is to define a subpopulation of cells called "marginal populations" by simultaneously detecting two emission wavelengths of a single fluorescent dye, thereby detecting the intended Hematopoietic Stem Cells in the mouse bone marrow and in human umbilical cord blood.
Operation method
Detection and enrichment of hematopoietic stem cells by marginal group cell phenotyping
Principle
The basic principle of Hematopoietic Stem Cell Detection and Enrichment by Marginal Population Cell Phenotyping is to define a subpopulation of cells called "marginal populations" by simultaneously detecting two emission wavelengths of a single fluorescent dye, thereby detecting the intended Hematopoietic Stem Cells in the mouse bone marrow and in human umbilical cord blood.
Materials and Instruments
Equipment: flow cytometer, scissors, bone biting forceps, syringes, needles, forceps, 80um filters/screens, sterilized tissue culture vessels, hemocytometer, water bath, cryo-centrifuge. Move The experimental steps for detection and enrichment of hematopoietic stem cells by marginal group cell phenotyping were as follows: Painlessly execute the mice according to the prescribed method. A. Sterilize with 70% ethanol and place on the laboratory bench. B. Cut a transverse incision in the middle of the abdomen with sterilized surgical instruments and remove the skin of the hind limbs and buttocks. C. Free the hind limb from the marrow joint, remove the paw, and place it in a vessel with buffer. D. Tick off the muscle tissue on the femur and tibia and transfer to a new vessel containing buffer. E. Separate the femur and tibia by removing the ends of the bones. F. Transfer the femur and tibia into a new vessel containing buffer and gently rinse out the marrow from the ends of the bone with 4 mL of buffer. G. The cell suspension is aspirated into a 10 mL tube and the vessel is washed with new buffer to ensure that all cells are recovered. H. The cell suspension is filtered through an 80 pjn sieve to remove cell clumps.
Reagents:
①70% ethanol;
② Buffer: phosphate buffer solution (PBS) or Hank's balanced salt solution (HBSS) and 2% (v/v) fetal bovine serum (FBS);
(iii) Medium: high sugar DMEM, containing 2% (v/v) FBS and 10 mM HEPES;
(iv) Solution for isolation of single nucleated cells from human umbilical cord blood: anticoagulant
⑤ Erythrocyte lysate: 154 mM ammonium chloride, 10 mM sodium or potassium carbonate, and 0.082 mM tetrasodium EDTA.
⑥ Hoechst 33342: Prepare 1 mg/mL storage solution and dissolve in distilled water.
(7) Verapamil (Sigma): Prepare a 5 mM stock solution in distilled water and store at 4℃.
(8) Fumonisin C (FTC): Prepare 10 mM storage solution with DMS0 and store at -20℃.
(9) propylene iodide (PI): deionized water was prepared into lmg/mL storage solution, stored at 4 ℃ away from light.
A. Dilute anticoagulated cord blood 1:3 with room temperature, sterile PBS containing 0.6% ACD-A or 2 mM EDTA.
B. Place 12 mL of Ficoll-Paque in a 50 mL tube and slowly add 35 mL of diluted cord blood on top.
C. Centrifuge at 20°C, 375 g, horizontal rotor for 30 min.
D. Aspirate the upper clear liquid layer to the mononuclear cell layer (a thin white layer at the junction).
E. Using a 10 mL pipette, transfer the cell layer to a 50 mL tube (approximately 20 mL per tube).
F. Top up each tube to 50 mL with PBS containing 0.6% ACD-A or 2 mM EDTA at room temperature and centrifuge at 375 g for 15 min at 20°C. G. Add 10 mL of erythrocytes to the tube.
G. Resuspend cells with 10 mL of erythrocyte lysate (mix well) and top up to 50 mL.
H. Allow to stand at room temperature for l0 min, then centrifuge at 20°C, 375 g for 15 min.
I. Wash the cells with 20 mL of room temperature PBS and centrifuge at 375 g for 15 min, then resuspend the cells with pre-warmed culture medium.
(iii) Staining of Hoechst 33342 cells.A. Set the water bath to 37°C and preheat the culture medium.
B. Count the number of nucleated cells and suspend the cells at 1x106/mL with culture medium.
C. Add Hoechst 33342 stock solution to the cell suspension at a final concentration of 5ug/mL.
D. Place the cell/dye suspension in an appropriate tube and submerge in a water bath. Use 5 mL tubes for volumes 1-3 mL, 15 mL tubes for volumes 4-10 mL, and 50 ml tubes for larger volumes. Be sure to completely submerge the top layer of the cell suspension in the water bath. Be sure to keep the top of the cell suspension completely submerged in the water bath to ensure that the sample is heated uniformly at 37°C.
E. Water bath for 90 min, mixing every 20 min with gentle inversion to avoid settling and clumping of cells.
F. Centrifuge at 375 g for 6 min at 4°C (pre-cool the rotor of the centrifuge) and resuspend the cells in an appropriate amount of pre-cooled buffer.
(D) Detection of SP cells by flow cytometryA. Install a 450/20 bandpass filter for detecting Hoechst blue, a 675 long bandpass filter for detecting Hoechst red, and a 610 short-wave bandpass dichroic prism for distinguishing between blue and red light signals.
B. UV-excited PI fluorescence is also detected by the 675 long bandpass filter. However, PI-positive dead cells fluoresce with high intensity and can be easily distinguished from Hoechst red on live cells.
C. Create the following two-dimensional point plots (x- and y-axes) with linear patterns for all parameters: forward scattered light (FSC) and side scattered light (SSC). Hoechst Red and Hoechst Blue.
D. Samples were stored at low temperature and protected from light during analysis.
E. On the sample, observe the FSC and SSC dot plots simultaneously and adjust the voltage of the two parameters until all cells are displayed in the two-dimensional dot plot.
F. Adjust the voltage of the Hoechst Red and Hoechst Blue parameters so that cells with strong Hoechst 33342 coloration are visible in both 2D dot plots. Continue to increase the voltage so that the weakly colored cells of the Hoechst 33342 are shown in the lower left of the dot plot.
G. Harvest 100,000 to 500,000 cells.
H. The SP population is shown on the nonlinear dot plots of Hoechst Red and Hoechst Blue. SP cells are the discrete population on the left side of the dot plot, which fluoresces at low intensities on both emission waves.
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