Abstract
Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during the S-phase of the cell cycle. As such, BrdU incorporation can be used to quantify the number of cells that are in S-phase in the time period during which BrdU is available. The following protocol describes an in vivo BrdU incorporation assay as a measure of cell proliferation in adult murine hematopioetic stem cells (HSCs). Specifically, BrdU incorporation was analyzed for long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, Lin-Sca-1+c-Kit+CD34+CD135+) population.
Materials and Reagents
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Mouse
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BrdU
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RPMI1640
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Fetal Bovine Serum (FBS)
-
Potassium bicarbonate
-
Ammonium chloride
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EDTA
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BSA
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Lineage Cell Depletion Kit (including Biotin-Antibody Cocktail and Anti-Biotin MicroBeads) (Miltenyi Biotec, catalog number: 130-090-858 )
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BD Cytofix/Cytoperm Buffer (Becton, Dickinson and Company, catalog number: 554714 )
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BD Perm/Wash Buffer
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DNase (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619 )
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DPBS without calcium, magnesium (diluted from 10x DPBS) (Hyclone, catalog number: SH 30258.01 )
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Antibodies:
PE-conjugated-Sca-I (Becton, Dickinson and Company, catalog number: 553336 )
APC-conjugated-c-Kit (Becton, Dickinson and Company, catalog number: 553356 )
PerCP-eFluor 710-conjugated-CD135 (eBioscience, catalog number: 46-1351-80 )
eFluor 450-conjugated CD34 (eBioscience, catalog number: 48-0341-80 )
BrdU-FITC (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619)
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Buffer A (see Recipes)
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Red blood cell lysis buffer (see Recipes)
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Staining buffer (see Recipes)
Equipment
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Small scissors and forceps
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60 mm tissue culture dish
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23 G needle
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3 cc syringe
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15 ml centrifuge tube
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Cell strainer (Becton, Dickinson and Company, catalog number: 352340 )
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Hemacytometer
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MACS MS column (Miltenyi Biotec, catalog number: 130-042-201 )
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MiniMASC separator (Miltenyi Biotec, catalog number: 130-042-102 )
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Centrifuge
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BD LSRFortessa Analytical Flow Cytometer
Procedure
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Animal injection
i.p. Injection of BrdU (10 mg/ml) 100 μl to 8-12 weeks old mouse (50 μg/g BW, so 5 μl/g of BW), after 6 h, inject the second dose. 2 h post 2nd injection, euthanize the mice using CO2 method followed by cervical dislocation, obtain the bone marrow (BM) cells from 2 tibias and 2 femurs. Two injections ensure that BrdU can incorporate to DNA of both slow and quick turnover cells.
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Obtain total bone marrow (BM) cells
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Use small scissors and forceps, dissect out femurs and tibias from
mice and place them in a 60 mm tissue culture dish containing 6 ml
ice-cold RPMI1640 with 5% heat inactivated FBS. Use Kimwipe tissue to
remove muscle and other tissues. Cut off both ends of each bone shaft in
the dish.
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Connect the end of the bone with 23 G needle on 3
cc syringe, flush out bone marrow with RPMI1640 with 5% heat
inactivated FBS into the dish. Disaggregate bone marrow tissues by
repeated aspirations using the same needle. Transfer the cell suspension
to 15 ml centrifuge tube.
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Spin down the cells at 350 x g for 5 min at room temperature, remove the supernatant, resuspend the
cells in 1 ml of room temperature red blood cell lysis buffer and
incubate at room temperature for 5 min, then add 5-10 ml of RPMI 1640
with 5% heat inactivated FBS.
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Pass the cells through a cell
strainer. Collect the flow through to a new tube. Take an aliquot and
count the cells in a hemacytometer. Spin down at 350 x g for 5 min at
room temperature. Remove the supernatant; the cell pellet should not
contain any red color. Disaggregate the cell pellet and wash the cells
one time with buffer A, spin down at 350 x g for 5 min at room
temperature.
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Lineage cell staining (follow the mouse Lineage Cell Depletion Kit)
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Resuspend the total BM cells in buffer A (40 μl/107 cells).
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Add
Biotin-Antibody Cocktail (10 μl/107 cells) to stain the lineage
differentiated cells. Cocktail of biotin-conjugated monoclonal
antibodies contains anti-CD5, anti-CD45R(B220), anti-CD11b,
anti-Gr-1(Ly-6G/C), anti-Neutrophil (7/4) and anti-Ter-119.
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Mix well and incubate for 10 min at 4 °C.
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Add additional buffer A in media (30 μl/107 cells) then add Anti-Biotin MicroBeads (20 μl/107 cells, provided in Lineage Cell Depletion Kit).
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Mix well and incubate for 15 min at 4 °C.
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Wash cell by adding 2 ml of buffer A. Centrifuge at 300 x g for 10 min at room temperature.
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Remove the supernatant and resuspend the pellet in 0.5 ml of buffer A.
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Lineage depleation
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Place MACS MS column in MiniMASC separator.
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Prepare column by rinsing with 0.5 ml buffer A.
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Apply cell suspension onto the column. Allow the cells to pass through and collect flow through as Lin- fraction.
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Wash column 3 times with buffer A (0.5 ml/each), wash each time once the column reservoir is empty.
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Collect all the elute (Lin-) in one tube.
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Count the Lin- cells, aliquot 1.5 x 106 cells to a new tube.
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Add 2 times more staining buffer to the cell suspension.
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Centrifuge the cells at 350 x g for 5 min.
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Stain the Lin- cells with surface antigens:
Test
|
Total
(μl)
|
Percp-eFluor 710
|
eFluor 450
|
PE
|
APC
|
Staining
buffer
|
CD135
|
CD34
|
Sca-I
|
CD117
(c-kit)
|
1
|
75
|
1
|
2
|
0.5
|
0.5
|
71
|
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Stain each test sample per 1.5 x 106 cells/75 μl buffer, make antibody mix as following, for more samples, increase antibody amount and staining buffer proportionally.
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Add 75 μl of antibody mix to the cell pellet. Incubate cells with antibodies for 15 minutes at room temperature (protected from light).
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Wash one time with staining buffer. Spin down for 5 minutes at 350 x g, and discard the supernatant.
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Fix and permeabilize the cells
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Resuspend the cells in 100 μl of BD Cytofix/Cytoperm Buffer per tube.
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Incubate the cells for 15 to 30 minutes at room temperature or on ice.
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Wash the cells with 1 ml of 1x BD Perm/Wash Buffer (dilute the 10x buffer with deionized H2O). Centrifuge at 350 x g for 5 minutes at room temperature, and discard the supernatant.
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Enhance the permeabilization:
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Resuspend the cells in 100 μl of BD Cytoperm Permeabilization Buffer Plus per tube. This reagent is specially formulated for the BrdU Flow kit and is used as a staining enhancer and secondary permeabilization reagent.
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Incubate the cells for 10 minutes on ice.
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Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
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Re-fix cells after secondary permeabilization:
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Resuspend the cells in 100 μl of BD Cytofix/Cytoperm Buffer per tube.
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Incubate the cells for 5 minutes at room temperature or on ice.
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Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
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Treat with DNase to expose incorporated BrdU
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Resuspend the cells in 100 μl of diluted DNase (diluted to 300 μg/ml in DPBS) per tube, (i.e. 30 μg of DNase/106 cells).
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Incubate cells for 1 hour at 37 °C.
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Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
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BrdU intracellular antigens staining
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Make diluted BrdU antibody (1 μl to 50 μl/sample in BD Perm/Wash Buffer).
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Incubate the cells for 20 minutes at room temperature.
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Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 4c).
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Resuspend the cells in 0.3 ml of staining buffer and perform flow cytometry analysis. Samples can be stored overnight at 4 °C, protected from light, prior to analysis by flow cytometry.
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Flow cytometry was performed on BD LSRFortessa Analytical Flow Cytometer with the following gating strategy (Figure 1).

Figure 1. Gating strategy to analyze BrdU incorporation in LT-HSCs. A. BM Lin- cells were labeled with PE-Scal-I and APC-c-Kit antibodies and analyzed by flow cytometry. B. Lin-/Scal-I+/c-Kit+ (LSK) cells were gated as showed in A and the LSK cells were further analyzed with eFluor 450-CD34 and PerCP-eFluor 710-CD135 staining. Long-term HSCs (LT-HSCs, shown as Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, shown as Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, shown as Lin-Sca-1+c-Kit+CD34+CD135+) were separated as indicated. C. BrdU incorporation was further analyzed for each cell population. Data shown are LT-HSCs population analyzed for BrdU staining.
Recipes
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Buffer A
DPBS, pH 7.2 supplemented with 0.5% BSA and 2 mM EDTA
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Red blood cell lysis buffer
155 mM potassium bicarbonate
10 mM Ammonium chloride
0.1 mM of EDTA, pH = 7.4
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Staining buffer
DPBS, pH 7.2 supplememted with 0.5% BSA and 0.09% sodium azide
Acknowledgments
This protocol is adapted from An et al. (2013).
References
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An, N., Lin, Y. W., Mahajan, S., Kellner, J. N., Wang, Y., Li, Z., Kraft, A. S. and Kang, Y. (2013). Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells. Stem Cells 31(6): 1202-1212.
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: An, N. and Kang, Y. (2013).
In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells.
Bio-protocol 3(21): e960. DOI:
10.21769/BioProtoc.960.