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Daniel Tenen

Hideyo Hirai

Pu Zhang

Tajhal Dayaram

Christopher Hetherington

Shin-ichi Mizuno

Jiro Imanishi

Koichi Akashi

Cell staining for sorting of hematopoietic stem cells \(HSC) and myeloid progenitors and isolating RNA from sorted cells

The combination of prospective identification/isolation of bone marrow progenitors and quantitative RT-PCR is a powerful tool to understand the molecular mechanism underlying hematopoiesis. Here, we described our standard procedures of the murine myeloid progenitor staining for fluorescence activated cells sorting \(FACS) and RNA purification methods.

**Reagents** 1&#xD7;PBS/2%FCS \(should be sterile if necessary)


ACK buffer \(41.45g NH4Cl, 5g KHCO3, 0.18g EDTA, H2O to 5 liters, store at 4&#xB0;C, filter to sterilize, if necessary).


Propidium iodide \(PI, Sigma), make 500&#xD7; stock solution \(1.0mg/ml) in a fume hood.





**Antibodies** \(The amount of the antibodies provided in this protocol is just an example. It is necessary to titrate all the antibodies every time when using a new batch even if it has the same catalog number.) 


Tricolor \(TC=phycoerythrin PE-Cy5) conjugated rat antibodies specific for CD3, CD4, CD8, CD19, B220 and Gr-1 \(Caltag).


Allophycocyanin \(APC)-conjugated anti-c-kit \(BD Pharmingen).


FITC-conjugated anti-CD34 \(BD Pharmingen).


Biotinylated anti-Sca-1 \(BD Pharmingen).


PE-conjugated anti-Fc&#x3B3;RIII/II \(eBioscience). 


Strepavidin&#x2013;APC-Cy7 \(Caltag).


Anti B220 antibody conjugated to FITC, PE, APC \(BD Pharmingen), TC and APC-Cy7 \(Caltag).





RNeasy Micro kit \(Qiagen).


&#x3B2;-mercaptoethanol \(&#x3B2;-ME, Sigma).

5ml polypropylene tube with snap cap \(12 &#xD7; 75 mm).


50ml polypropylene conical tube. 


Dynabeads \(Sheep anti-Rat IgG) and magnet rack \(Dynal Biotech).


Nylon mesh \(40-70 &#x3BC;m pore size).

1. Harvest bone marrow \(BM) cells and spleen cells and resuspend them in 1&#xD7;PBS/2%FCS in 50 ml conical tubes.
 2. Spin down at 1300 rpm for 5 minutes.
 3. Discard liquid completely.
 4. Add 500 &#x3BC;l of ACK buffer, pipet and wait on ice or RT for 1-5 minutes \(until the solution is clear).
 5. Add 40 ml of 1&#xD7;PBS2%FCS and mix.
 6. Spin down at 1300 rpm for 5 minutes.
 7. Resuspend cells as follows. \(It is advised to use nylon mesh to filter the single cell suspensions before and after the staining to avoid clogging the FACS machine). 
 BM cells from 1 mouse in 100 &#x3BC;l of 1&#xD7;PBS/2%FCS \(increase the volume accordingly to the number of the mouse) in a 5 ml tube. 


Spleen cells from 1 mouse in 350 &#x3BC;l of 1&#xD7;PBS/2%FCS.





**BM cells**


1. Add 3&#x3BC;l of anti CD3-TC Ab, 2&#x3BC;l of anti CD4-TC antibody, 2&#x3BC;l anti CD8a-TC Ab, 2&#x3BC;l anti CD19-TC Ab, 3&#x3BC;l of anti-Ly-6G \(Gr-1)-TC Ab, 3&#x3BC;l of anti-CD45R \(B220)-TC Ab.
 2. Mix and incubate for 30 minutes on ice in the dark.
 3. Wash the cells twice with 3ml of 1xPBS/2%FCS.
 4. Lineage depletion, if necessary \(if not, can skip this step and combine step 1 and 6).
 \(a) Resuspend BM cells in 100&#x3BC;l 1&#xD7;PBS/2%FCS and add 70&#x3BC;l of Dynabeads.


\(b) Rotate tube in dark at 4&#xB0;C for 30 minutes.


\(c) Add 1&#xD7;PBS/2%FCS to 4.5 ml and mix.


\(d) Put the tube onto Dynal magnetic rack and leave the tube on the rack for 2 miuntes at RT.


\(e) Transfer the supernatant containing the lineage negative cells to a new tube with a Pasteur pipet \(if leaving it overnight, put it in 15-20 ml of 1&#xD7;PBS/2%FCS at 4&#xB0;C in the dark.)


\(f) Spin down at 1300 rpm for 5 minutes.


5. Resuspend the BM cells in 100&#x3BC;l 1&#xD7;PBS/2%FCS.
 6. Add 5&#x3BC;l anti-CD34-FITC Ab, 3&#x3BC;l of anti-Fc&#x3B3;RIII/II-PE Ab, 2&#x3BC;l anti-CD117 \(c-kit)-APC Ab and 2&#x3BC;l anti-Sca-1 \(Ly-6A/E)-biotin Ab.
 7. Mix and incubate on ice for 30 minutes in dark.
 8. Wash the cells twice with 3 ml of 1&#xD7;PBS/2%FCS.
 9. Resuspend in 100&#x3BC;l 1&#xD7;PBS/2%FCS.
 10. Add 2 &#x3BC;l APC-Cy7 conjugate Streptavidin.
 11. Mix and incubate on ice for 30 minutes in dark.
 12. Wash the cells twice with 3 ml of 1&#xD7;PBS/2%FCS and resuspend the cells \(the best concentration is 5 &#xD7; 107/ml for MoFlo).
 13. Add propidium iodide to stain dead cells \(final concentration =2&#x3BC;g/ml).
 14. Store on ice with no light until the analysis/sorting.
 


**Spleen cells** \(we use spleen cells for single color staining FACS compensation controls)


1. Aliquot spleen cells to 7 tubes \(50&#x3BC;l each) and mark each tube as #1-#7.
 2. Add the following antibodies to each tube.
 tube #1 no addition


tube #2 add propidium idodide \(final concentration =2&#x3BC;g/ml)


tube #3 1&#x3BC;l of anti-B220-FITC


tube #4 1&#x3BC;l of anti-B220-PE


tube #5 1&#x3BC;l of anti-B220-APC


tube #6 1&#x3BC;l of anti-B220-APC-Cy7


tube #7 1&#x3BC;l of anti-B220-TC


3. Mix and incubate on ice for 30 minutes in dark.
 4. Wash the cells twice with 3ml of 1&#xD7;PBS/2%FCS.
 5. Resuspend cells in 500&#x3BC;l of 1&#xD7;PBS/2%FCS \(don&#x2019;t add PI except tube #2).
 


**RNA preparation**


1. Purify RNA using a RNeasy Micro Kit \(Qiagen). 
 2. Isolate HSC or myeloid progenitor cells \(CMP, GMP or MEP) using a DakoCytomation High Speed MoFlo Sorter and then resort directly into a microfuge tube containing 350&#x3BC;l of RLT buffer with &#x3B2;-ME and carrier RNA provided by the manufacturer. Double sorting is necessary to get reliable data.
 3. Lyse the cells by vortexing for 1 minute when the cell number is lower than 1 &#xD7; 105.
 4. Purify RNA according to the manufacturer&#x2019;s protocol \(Qiagen). We use a DNase treatment to avoid contamination with genomic DNA. 5. Add a DNaseI incubation mix \(10&#x3BC;l DNase I stock solution plus 70&#x3BC;l Buffer RDD provided by the manufacturer, Qiagen) onto a column and incubate for 15 min between the first and second treatments with RW1 buffer.

Akashi K, Traver D, Miyamoto T, Weissman IL. _Nature._ 2000 Mar 9; **404\(6774)**:193-197.


Iwasaki H, Somoza C, Shigematsu H, Duprez EA, Iwasaki-Arai J, Mizuno S, Arinobu Y, Geary K, Zhang P, Dayaram T, Fenyus ML, Elf S, Chan S, Kastner P, Huettner CS, Murray R, Tenen DG, Akashi K. _Blood._ 2005 Sep 1; **106\(5)**:1590-1600.

None

Cell staining for sorting of hematopoietic stem cells (HSC) and myeloid progenitors and isolating RNA from sorted cells

Method Article

Daniel Tenen, Hideyo Hirai, Pu Zhang, Tajhal Dayaram, Christopher Hetherington, Shin-ichi Mizuno, Jiro Imanishi, Koichi Akashi

Daniel Tenen

Corresponding Author

Hideyo Hirai

Pu Zhang

Tajhal Dayaram

Christopher Hetherington

Shin-ichi Mizuno

Jiro Imanishi

Koichi Akashi

DOI:

10.1038/nprot.2006.139

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Associated Publications

C/EBPbeta is required for 'emergency' granulopoiesis

by Hirai, H. et al.

Nature Immunology (18 April, 2006)

Method Article

Daniel Tenen, Hideyo Hirai, Pu Zhang, Tajhal Dayaram, Christopher Hetherington, Shin-ichi Mizuno, Jiro Imanishi, Koichi Akashi

Daniel Tenen

Corresponding Author

Hideyo Hirai

Pu Zhang

Tajhal Dayaram

Christopher Hetherington

Shin-ichi Mizuno

Jiro Imanishi

Koichi Akashi

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DOI:

10.1038/nprot.2006.139

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C/EBPbeta is required for 'emergency' granulopoiesis