Phenotypic and functional analysis of CD34NEG hematopoietic precursor cells mobilized from peripheral hematopoietic pools is an experiment to study the modulation of CD34 antigen expression during the proliferative state of primitive CD34NEG CD38NEG LINNEG cells and CD34+ CD38NEG LINNEG cells under serum-free culture conditions.
Principle
Peripheral Hematopoietic Pool Mobilized CD34NEG Hematopoietic Precursor Cells Phenotypic and Functional Analysis is performed by purifying quiescent primitive CD34+ and CD34NEG cells from cells mobilized from peripheral hematopoietic pools. Unlike other sources of CD34NEG cells, peripheral hematopoietic pool-mobilized cell-derived CD34NEG cells are easily grown in stroma-free cell cultures and are easy to analyze. CD34NEG CD38NEG LINNEG and CD34+CD38NEG LINNEG cells were obtained by cell sorting and purification using low-density single nucleated cell fractions after spectrum antigen clearance. Up- and down-regulation of CD34 antigen expression was observed when the cells were cultured in serum-free culture medium containing early activating cytokines for 40 h. The cells were then incubated in a serum-free culture medium for 10 h.
Operation method
Experimental phenotypic and functional analysis of CD34NEG hematopoietic precursor cells mobilized from peripheral hematopoietic pools
Principle
Peripheral Hematopoietic Pool Mobilized CD34NEG Hematopoietic Precursor Cells Phenotypic and Functional Analyses were performed by purifying quiescent primitive CD34+ and CD34NEG cells from cells mobilized from peripheral hematopoietic pools. Unlike other sources of CD34NEG cells, peripheral hematopoietic pool-mobilized cell-derived CD34NEG cells are easily grown in stroma-free cell cultures and are easy to analyze. CD34NEGCD38NEGLINNEG and CD34+CD38NEGLINNEG cells were obtained by cell sorting and purification using low-density single nucleated cell fractions after spectrum antigen clearance. Up- and down-regulation of CD34 antigen expression was observed when the cells were cultured in serum-free culture medium containing early activating cytokines for 40 h. The cells were then subjected to a series of cell sensitization tests.
Materials and Instruments
Equipment: Move The basic process of phenotypic and functional analysis of CD34NEG hematopoietic precursor cells mobilized from peripheral hematopoietic pools experiment can be divided into the following steps: A. Quickly place the samples in a 37 °C water bath. Slowly dilute the sample with an equal volume of cold IMDM containing 20% HIFBS and mix continuously, centrifuge at 300 g for 10 min at room temperature. B. Discard the supernatant and wash the cells with 30 ml of room temperature DNAzyme wash solution. Discard the supernatant and resuspend the cells evenly in 10 ml of DNAzyme wash solution. C. In a 15 ml tube, add the cell suspension to the surface of a 5 ml Ficoll-Paque isolate. Centrifuge at 300 g for 25 min at room temperature to collect low density MNC bands, place in a 50 ml tube, add DNA Enzyme Wash Solution, and centrifuge twice. Minimize cell loss by centrifugation at 400 g for 15 min at room temperature (use the same wash solution for fresh and frozen cells). D. Remove as much of the supernatant as possible, add 15 ml ammonium chloride-EDTA to the precipitate, mix well, and allow to stand at room temperature for 10 min to dissolve any remaining erythrocytes. Fill tubes with PBS and centrifuge at 300 g for 10 min at room temperature, wash with DNAase wash buffer, and remove a small portion for cell counting before the last wash (to determine the amount of supernatant to be added for the last wash). E. Resuspend the cells with DNAzyme Wash Solution to a concentration of 6 × 107/ml [the permissible cell concentration range for each column is (2-8) × 107/ml ].
① Green magnet (Catalog No. 11 055) and green magnetic holder (Catalog No. 11 056);
② 1.27 cm diameter separation column (Catalog No. 12 052) or 1.524 cm diameter separation column (Catalog No. 12 056);
③ Rainin Dynamax pump (RP-1 peristaltic pump, Rainin Instrument Co., Inc., Winburn, MA);
④ Fresh or frozen MOPB cells. Samples must contain cells ≥ 4x108MNC (a high starting cell count is beneficial for subsequent experiments);
⑤ The flow cytometer must be equipped with 2 lasers that emit 488 nm (FITC, PY, PE, and PE-CY5) and 633 nm (excitation of alpha-pyrocyanin, APC).
Reagents:
① Iscove's modified Dulbecco's culture medium (IMDM) (Carlsbad, CA, catalog number 12 440-053);
② Serum-free amplification medium (SFEM) (Vancouver, British Columbia, Canada, Catalog No. 9 650);
③ Heat-inactivated fetal bovine serum (HIFBS);
④ Cytokines: rhSCF, rhFlt3 Ligand (rhFL), rhIL-3, rhIL-6, and rh-TPO;
⑤ Sterile 10 × phosphate buffer solution (PBS), free of calcium and magnesium ions (Catalog No. 14 200-075), diluted to 1X with sterile distilled water;
⑥ DNA enzyme (bovine pancreatic deoxyribonuclease I) (St. Louis, MO, Catalog No. 2025);
(vii) Lymphocyte isolate (Piscataway, NJ);
(viii) Ammonium chloride-EDTA (Catalog No. 7,850);
⑨ Non-Fluorescent Antibodies for Immunomagnetic Separation from StemCell Technologies: Reagents for the Spectral Clearance of Magnetically Separated Cell Components (Human Progenitor Cell Enrichment Combination Antibody, Catalog No. 14 056) consisting of antibodies directed against Glycoprotein A (GlyA), CD2, CD3, CD14, CD16, CD19, CD24, CD56, and CD66b. CD66b, containing magnetic colloids, as well as the required CD41 tetrameric antibody complex (Catalog No. 14 050);
⑩ BD Biosciences (San Jose, CA) Fluorescent Antibodies: FITC-labeled antibodies: CD3, CD7, CD14, CD16, CD19, CD20, CD42a, and CD56 (CD7 and CD42a antibodies are included in the Spectrum Mix, Catalog No. 340 546), and in addition, CD38-APC, Ki67-FITC kit (BD Biosciences, #556 026) and appropriate isotype control antibodies;
⑪ Beckman-Coulter (Miami, FL) fluorescent antibodies: GlyA-FITC, CD34-PE-CY5 (clone 581, class III) and isotype control antibodies. The following PE-labeled antibodies are also required: GlyA, CD4, CD14, CD16, CD19, CD20, CD41, and CD56. 100 μl of each antibody is mixed into a single "LIN-PE" reagent, which is stored in a black vial at 4 ℃. For cell analysis, add 25 μl of the mixture to each tube, and the appropriate isotype control antibody is also required;
⑫ Cell Fixation/Cell Breaking; Kit (Catalog No. 554 714);
⑬ Penicillin + Streptomycin Solution, 10,000 U (Catalog No. 15 140-122);
⑭ Low Density Lipoproteins (LDLs) (Catalog # L2 139);
⑮ Immuno-Brite quality control beads (Catalog No. 6 603 473);
⑯ Paraformaldehyde (PFA) (Sigma-Aldrich);
⑰ Pyronin Y (PY) (Sigma-Aldrich);
⑱ Hoechst 33 342 (HO) (Sigma-Aldrich);
⑲ Overnight storage medium (OSM): 10 ml SFEM containing 100 μl of penicillin/streptomycin, 20 μl of LDL;
⑳ 10 ng/ml of flt3 ligand (FL), 10 ng/ml of stem cell factor (SCF);
㉑ DNA enzyme wash solution: 100 ml sterile PBS containing 1 mg DNA enzyme, 1% penicillin/streptomycin, 2% HIFBS. prepared fresh for use on the same day;
㉒ Supplemental SFEM: SFEM containing 1% bovine serum albumin, 10 μg/ml insulin, 200 μg/ml human transferrin (iron saturated), 10
M 2-mercaptoethanol
M 2-mercaptoethanol, 2 mM L-glutamate in IMDM supplemented with 50 ng/ml rhSCF, 50 ng/ml rh-FL, 12.5 ng/ml rhIL-3, 10 ng/ml rhIL-6, 100 ng/ml TPO and 1:200 LDL;
㉓ Paraformaldehyde (PFA): dilute to 1% concentration with 4% paraformaldehyde PBS solution monthly. Dilute an additional 2 to 5 times for cell fixation;
㉔ PBS Fetal Bovine Serum (PBSFB): 1 × PBS containing 2% HIFBS and 1% penicillin/streptomycin;
㉕ N-(α-hydroxyethyl)zine-N'-(α-ethanesulfonic acid) (HEPES) buffer: pH 7.2, Hank's equilibrium salt solution containing 20 mM HEPES, 1 g/L glucose, and 10% HIFBS, stored at 4 °C;
㉖ HO: storage solution of 1.6 mM, filtered and sterilized, stored at 4 °C protected from light. ㉖ HO: storage solution is 1.6 mM, filtered and sterilized, stored at 4 ℃, protected from light;
㉗ PY: 100 μg/ml of storage solution, filtered and sterilized, stored at 4 ℃ away from light. On the day of use of the dye: dilute the storage solution to 10 μg/ml with HEPES buffer and place on ice protected from light.
A. 1st Incubation: Add 100 μl Human Hematopoietic Progenitor Cell Enrichment Combination Antibody Reagent to each 1.0 ml of cell suspension. Also add anti-CD41 to the cell suspension to a concentration of 10 μl/ml. incubate on ice for 30 min.
B. 2nd Incubation: Without washing, add 60 μl of magnetic colloid per 1.0 ml of cell suspension. Then ice bath again for 30 min without washing the cells.
C. The isolation system is designed to enrich LINNEG cells by removing LIN+ cells and consists of a magnet, isolation column, holder and suitable suction pump. The green magnet can be used to gravity load or pump cells, and the other magnets are designed for gravity inflow of smaller numbers of cells or single or multiple pump separations. The manufacturer recommends a 1.27 cm column for 5 × 107 to 5 × 108 cells and a 1.524 cm column for 1 × 108 to 2 × 109 cells. Cell purity will be compromised if the column used is too small, and cell yields will be reduced if too large a column is used.
D. Inside the laminar flow ultra-clean bench, drop the separation column in the gap of the magnet (do not insert it from the front). Pre-prepare the separation system by slowly pumping sterile PBS from the top of the column toward the bottom until the column filling mass is completely submerged (1.27 cm column with the pump unit set to 1.5 or 1.524 cm column with the pump unit set to 3.0). Vigorously tap the side of the column to remove air bubbles. Allow sterile PBS (15 ml for 1.27 cm columns, 25 ml for 1.524 cm columns) to flow from the top of the column and collect the waste solution in a waste tube. Do not allow the level of PBS to drop below the filling material. Replace the waste tube with a sterile collection tube (50 ml centrifuge tube). Add the colloidal suspension of cells to the top of the separation column at a pump speed of 5.0 for a 1.27 cm column (10.0 for a 1.524 cm column). LINNEG cells will appear in the column washout.
E. To capture all LINNEG cells, flow the column with 3 times the column volume of DNAzyme eluent and collect the column eluate. Combine the column eluates for viable cell counting.
F. The combined column eluates were centrifuged at 300 g for 10 min at room temperature, the supernatant was removed, and the cells were resuspended in 10 ml of OSM. The cells were incubated overnight in T25 culture flasks (without any treatment to increase the cell adhesion capacity) at 37 °C with 5% CO2 humidity in an incubator. Under these conditions, cell activity was maintained and cell proliferation was not stimulated.
(iii) Isolation of primary CD34NEG and CD34+ cellsA. After overnight incubation, count the number of viable cells in the cell suspension obtained in the bottles (an average of 107 cells can be obtained from the starting 4 × 108 MoPB specimens).
B. Expansion of ex-LIN cells. These cells will be used as positive controls in subsequent experiments (see Subheading 3.5.1).
C. Removed LIN cells harvested from T25 culture flasks are placed in 15 ml test tubes containing sterile PBS and centrifuged at 300 g for 10 min at room temperature.
D. Resuspend the cells with 0.5-1.0 ml of PBSFB, and remove the portion in a test tube for antibody labeling. According to the laboratory's own experience, use the remaining cells to establish a sorting control. Add 2.0 ml of PBS to each tube and centrifuge at 300 g at 4 ℃ for 10 min. 200 μl of supernatant is retained, the rest is discarded, and the cells are resuspended in the supernatant.
E. In this step, the cells to be sorted are labeled with antibodies against CD34, CD38, and LIN antigens. A commercial LIN antibody combination of FITC-labeled anti-CD3, CD14, CD16, CD19, CD20, and CD56 is added to the FITC-labeled anti-CD42a, CD7, and GlyA antibodies to serve as the labeled lineage antigen antibody combination. For every 1 × 106 cells, 12 μl of LIN-FITC combination and 15 μl each of GlyA-FITC, CD7-FITC and CD38-APC were added, and 10 μl of CD34-PE-CY5 and CD42a-FITC were added. appropriate isotype control was set, and the cells were incubated for 30 min on ice and protected from light at 4 ℃, and the cells were washed twice with PBSFB. Resuspend the cells in PBSFB to the appropriate cell concentration for sorting. Keep the cells away from light and cold.
F. After completing the internal quality control operation of the flow cytometer, the isotype control specimen is uploaded. Sample with a small number of labeled cells to gate the sorting of CD34NEGCD38NEGLINNEG cells and CD34+CD38NEGLINNEG cells. For the most efficient purification of cells free of CD34, CD38, and LIN antigen, select a conservative gate that contains the 1/3 to 1/2 of the antigen-negative cell population with the weakest fluorescence intensity. Looser gating increases yield but decreases cell purity.
G. Sort the cell suspension with LIN removed to obtain CD34NEGCD38NEGLINNEG cells and CD34+CD38NEGLINNEG cell fractions. Keep the pre-sorted cell suspension and the 2 post-sorted cell fractions at low temperature throughout the procedure.
H. Count the live cells after sorting.
I. Take the isolated CD34+ and CD34NEG cell fractions from the step 10 experiment. Approximately 1 × 104 cells. Transfer the remaining purified cells to a 24-well plate supplemented with SFEM and incubate at 37 °C with 5% CO2 humidity. The initial cell culture concentration should be <5 × 104/ml.
J. Analyze the freshly sorted fractions by PY incorporation or Ki67 expression. Because freshly sorted cells are in stationary phase, they are part of the PYLOW or Ki67NEG cell population. Specimens can also be restained with CD34, CD38 and LIN to analyze cell purity.
K. When sorting is complete, without changing any voltage settings, resample Immuno-Brite beads #2 or #3. This will provide a reference fluorescence intensity for later Ki67 analysis.
(iv) Pre- and post-sorted cell culture immunostaining and proliferation status analysisA. If cells are cultured for 2 d or longer, the number of cells available is low and highly variable. If possible, remove (1~4) x 104 cells from cultured CD34NEG cells, 2 × 105 cells from CD34+ cells, and 4 × 105 cells from cell fractions with LIN removed. Each was washed with PBS and cells were resuspended with PBS.
B. Labeling A~D4 tubes for cultured cells from which LIN antigen has been removed serve as control cells. Also labeled: 4 tubes for E+, ENEG, F+, FNEG (2 each for CD34+ cells and CD34NEG cells, respectively) and 1 G tube (Immu-no-Brite beads) for a total of 9 tubes:
A = unstained specimens for determination of autofluorescence.
B = Ki67-FITC alone (positive control).
C = LIN-PE alone (positive control).
D = 34-PE-CY5 alone (positive control).
E+ and ENEG = isotype control-PE-CY5, isotype control-APC, isotype control-PE and isotype control-FITC ( E+ for CD34+ cultured cells, ENEG for CD34NEG cultured cells).
F+ and FNEG = Ki67-FITC, LIN-PE, 34-PE-CY5, 38-APC assay antibodies ( F+ for CD34+ cultured cells, FNEG for CD34NEG cultured cells).
G = Immuno-Brite beads.
C. 1 × 105 cells with the LIN component removed were added to tubes A, B, C, and D. For tubes E and F, 1 × 105 cells were added according to the laboratory. For tubes E and F, use the smallest amount of cells possible according to the laboratory's internal reference standards. For example, add ≥ 1 × 104 cultured CD34NEGCD38NEGLINNEG cells and ≥ 1 × 104 cultured CD34NEGCD38NEGLINNEG cells to E+ and ENEG tubes. Repeat the above with the same amount and type of cells in F+ and FNEG tubes.
D. For tubes A and B, wash the cells, resuspend the cells with 0.5-1.0 ml of PBSFB, and store in an ice bath protected from light.
E. For tubes C~F, stain with labeled monoclonal antibodies as per in-house laboratory standards for extracellular antigens. tubes C and D are washed for the last time and the cells are resuspended with 0.5~1.0 ml of PBSFB. Store in an ice bath. E and F (and B) tubes go directly to step F after the last 1 wash.
F. Fixation and rupture of cells in tubes B, E+, ENEG, F+, and FNEG for Ki67 staining was performed as follows:
G. Resuspend washed cells in tubes B, E+, ENEG, F+ and FNEG with 250 μl of cell fixation/film-breaking solution. Incubate for 20 min at 4 ℃, protected from light, and dilute the membrane-breaking/washing buffer 1:10 with distilled water during this 20-min period.
H. Wash tubes B, E+, ENEG, F+ and FNEG twice with diluted membrane-breaking/washing buffer at 4 ℃. After the 2nd centrifugation, remove the supernatant and resuspend the cells with only 50 μl of the remaining Rupture/Wash Buffer.
I. Add 20 μl of Ki67-FITC (experimental tube) into tube B and 2 tubes of F. Add 10 μl of isotype control-FITC (control tube) into 2 tubes of E. Incubate for 30 min on ice in the dark. Incubate for 30 min on ice in the dark.
J. Wash tubes B, E+, ENEG, F+ and FNEG twice with diluted membrane-breaking/washing buffer, and resuspend the cells with PBSFB for flow analysis. During intracellular staining, the reagent instructions require that the cells be kept in saponin-containing (membrane-breaking/washing) buffer.
K. Samples were assayed within 24 h. The Calibrate flow cytometer was first set to the highest channel values as described previously using the test tube G Immuno-Brite beads.
L. CD34, CD38, LIN, and PY-expressing cells were stained.
M. Day of use: Dilute HO/HEPES buffer: 10 μl HO stock solution added to 10 ml HEPES buffer. Dilute PY: 10 μl stock solution PY added to 90 μl HEPES buffer.
N. This section describes the collection and sorting of cells. Because of the obvious procedural differences from the method described in Subheading 3.5.2, the 4 cells are divided into Samples 1 to 4. As mentioned above, the number of cells available is small and variable.
O. Cells were cultured for 2 d or more, and (1~4) × 104 cells were removed from CD34NEG cell cultures (as "Sample 1"), 2 × 105 cells were removed from CD34+ cell cultures (as "Sample 2"), and 1 × 105 cells were removed from LIN-removed cultures (as "Sample 3"). Wash each tube with PBS and resuspend the cells with 1.5 mL of diluted HO/HEPES buffer. 3 tubes were capped, not screwed down, and immediately placed in an incubator at 37 ℃ with 5% CO2 humidity for 45 min. This is the first step for PY staining (the next step is step 6 below).
P. Remove 3 × 105 cells (as Sample 4) from the cultured removed LIN fraction, add to 2 ml volume with SFEM and store on ice.
Q. Label 4 tubes A to D for the determination of control cells in the LIN removal culture, and 4 tubes E+, ENEG, F+ and FNEG (2 for CD34+ cells and 2 for CD34NEG cells). A total of 8 tubes:
A = unstained cells (autofluorescence assay).
B = PY alone (positive control, PY has no substantial negative control).
C = Lin-FITC alone (positive control).
D = CD34- PE-CY5 (positive control).
E+ and ENEG = PY and isotype control-PE-CY5, isotype control-APC, and isotype control-FITC. ( E+ was used for CD34+ cultured cells, EN for CD34NEG cultured cells).
F+ and FNEG = PY and experimental LIN-FITC, CD34- PE-CY5, CD38- APC assay antibodies ( F+ for CD34+ cultured cells, FNEG for CD34NEG cultured cells).
R. Samples 1~3 After the initial 45 min incubation at 37 ℃, add 15 μl of diluted PY (final concentration of 0.1 μg/ml) to each tube. Put the lid back on, do not screw it on tightly, and immediately put it back to the incubator at 37 ℃ with 5% CO2 humidity for another 45 min.
S. Remove samples 1~3 from the incubator so that there are now 4 groups of cells: 3 stained with PY (steps 3 and 6) and sample 4 on ice (step 4). Divide these 4 samples into 8 test tubes.
T. For Sample 1, remove 1/3 of the CD34NEG cells and transfer to ENC tubes and add 2/3 to FNEG tubes. Cells were washed twice with 1.5 ml HEPES buffer. Resuspend cells with 200 μl PBS.
For U. Sample 2, remove 1/2 CD34+ cells and add to E+ tube and 1/2 to F tube. Wash cells twice with 1.5 ml HEPES buffer. Resuspend cells with 200 μl PBS.
V. Sample 3, wash cells twice with 1.5 ml HEPES buffer. Resuspend cells with 1.0 ml PBSFB. Place on ice, protected from light. This completes all steps for tube B.
W. Sample 4, divide the cells equally into tubes A, C and D and wash the cells twice with PBS. Resuspend cell A with 1.0 ml PBSFB on ice. Resuspend cells C and D with 200 μl of PBS so that tubes C, D, E+, ENEG, F+ and FNEG cells are ready for extracellular antigen staining.
X. Stain tubes C, D, E+, ENEG, F+, and FNEG cells with labeled LIN-FITC, CD34- PE-CY5, CD38- APC, or appropriate isotype control monoclonal antibody according to the standard method for staining extracellular antigens.
Y. Analyze on-line or after addition of 1% paraformaldehyde.
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