National Institutes of Health grant R01 CA149976 (N.A.S) supported this work.

All procedures must be performed in accordance with institutional animal use and care guidelines. The procedure was developed for the analysis of murine hematopoietic stem and progenitor cells (HSCs and HPs), but can be adapted for the analysis of other cell populations.
1. Flow Cytometry Isolation of Murine HSCs and HPs
<ol>
	<li>Harvest murine bone marrow cells as described in the literature6. 
	NOTE: In data presented in Figure 1 and Figure 2, bone marrow was harvested from C57BL/6J mice.</li>
	<li>Perform lineage depletion of bone marrow cells using a mouse lineage depletion kit, following the manufacturer&#39;s instructions.</li>
	<li>Incubate the cells with antibodies against the cell surface markers of interest as described7. In the experiments shown in Figure 1 and Figure 2, antibodies to Sca-1, Kit, Flt3, and lineage (Lin) markers Mac1, Gr1, CD3e, B220, and Ter119 are used.</li>
	<li>Sort 2,000 - 20,000 Lin- Sca1+Kit+ Flt3- cells (HSCs) or Lin-Sca1-Kit+ cells (HPs) into 1.5 mL Eppendorf tubes containing 0.1 mL of phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS). 
	NOTE: For the data shown in Figure 1 and Figure 2, cells were sorted using a Flow Cytometer with a 70 &#181;M nozzle. The maximum collection volume is 1.4 mL.</li>
</ol>
2. Sample Preparation
NOTE: This step is critical. Process the sample very carefully. When removing the supernatant, be very careful not to disturb the cell pellet.
<ol>
	<li>Centrifuge the 1.5 mL tubes containing sorted cells in a swinging bucket rotor at 4 &#176;C, 500 x g, for 5 min. Remove all but approximately 100 &#181;L of the supernatant from the cell pellet very gently using a pipette and a 20 &#181;L pipet tip. Do not disturb the pellet.
	<ol>
		<li>If the sample contains fewer than 5,000 cells, and the total volume of the sorted sample is less than 0.2 mL, transfer the cells first to low binding 0.2 mL PCR tubes before centrifugation.</li>
		<li>If sample contains fewer than 5,000 cells and volume is more than 0.2 mL, spin the cells down with a centrifuge at 4 &#176;C, 500 x g, for 5 min, and remove all but 200 &#181;L of the supernatant. Re-suspend the pelleted cells in the remaining 200 &#181;L of supernatant, then transfer the cells to the 0.2 mL PCR tubes and spin down again. Remove all but 20 - 30 &#181;L from the pellet after the second spin and re-suspend cells.</li>
	</ol>
	</li>
	<li>Resuspend the cells in the supernatant left in the tube. If necessary,add additional PBS in 2% FBS/PBS (you can also use 2% bovine serum albumin (BSA)/PBS, or PBS alone) to obtain a concentration 5 x104 to 5 x 105 cells/mL. 
	NOTE: Use the cell counts collected by cytometry to estimate the volume used to suspend cells.</li>
	<li>Use 2 - 5 &#181;L of the re-suspended cells to determine an accurate cell concentration using an automated cell counter (following the manufacturer&#39;s instructions) or a hemocytometer9.</li>
	<li>Transfer a volume of re-suspended cells containing the desired number of cells to new 1.5 mL tubes. For the experiment in Figure 1, 2,000, 1,000, or 500 HSCs or HPs were transferred. The desired number of cells depends on the strength of the signal obtained by Western blot, and is determined empirically. Perform the transfer using a 20 &#181;L or 100 &#181;L Eppendorf pipette tip.</li>
	<li>Centrifuge the cells in a swinging bucket rotor at 4 &#176;C, 500 x g, for 5 min.</li>
	<li>To generate 100x stock solutions, dissolve proteasome and phosphatase inhibitors in DMSO according to the manufacturer&#39;s instructions.</li>
	<li>Prepare 2x Laemmli sample buffer by diluting the 4x Laemmli sample buffer provided by the manufacturer with an equivalent amount of distilled water. Add an appropriate amount of the 100x stock of proteasome and phosphatase inhibitors to obtain a final concentration of 2x inhibitors in 2x Laemmli sample buffer. 
	NOTE: The 2x Laemmli sample buffer can be made in advance, but the proteasome and phosphatase inhibitors should be added immediately before adding the 2x Laemmli sample buffer (plus inhibitors) to the cell pellet to lyse the cells, as described in the following step.</li>
	<li>Carefully remove a portion of the supernatant from the cell pellet, and to the supernatant remaining in the tube with the cell pellet, add an equal volume of 2x Laemmli sample buffer plus proteasome and phosphatase inhibitors to achieve a final concentration of 500 cells/&#181;L in 1x Laemmli sample buffer. 
	NOTE: For example, if you transfer 2,000 cells in a total volume of 20 &#181;L to a tube in Step 2.5, after centrifuging the cells (Step 2.6) you should remove 18 &#181;L of the supernatant from the pellet, and to the 2 &#181;L of supernatant that is remaining add an equal volume (2 &#181;L) of 2x Laemmli sample buffer to achieve a final concentration of 500 cells/&#181;L in 1x Laemmli sample buffer.</li>
	<li>Resuspend the pellet to generate the lysate as soon as possible. At this point, the samples can be immediately electrophoresed through SDS-PAGE gels, or can be snap frozen in liquid N2 and stored at -80 &#176;C for future use.</li>
</ol>
3. Electrophoresis
<ol>
	<li>Heat the lysates in a heat block at 95 &#176;C or in boiling water for 5 min.</li>
	<li>Electrophorese 1 - 40 &#181;L of the lysates (containing from 500 up to 20,000 cell equivalents) through SDS-PAGE gels at 100V using standard protocols10. 
	NOTE: The volume of lysate loaded into the gel is determined by the number of cells needed to generate the desirable signal, and will depend on the abundance of the protein of interest, and the quality of the antibody. The data in Figure 1 were obtained with 2,000, 1,000, and 500 cell equivalents of lysate. The width of the well should be in the range of 3 mm to 1.5 mm. 1.5 mm teeth can be cut from a commercially available comb with wider teeth using scissors.</li>
</ol>
4. Transfer and Block
NOTE: Perform a Western blot following standard protocols11. The steps are briefly outlined here:
<ol>
	<li>Pretreat a polyvinylidene difluoride (PVDF) membrane with methanol for 10 s, then wash the membrane briefly with distilled water.</li>
	<li>Transfer the protein from the SDS-PAGE gel to the membrane following the instructions in the manual provided by the manufacturer of the transfer apparatus.</li>
	<li>Following transfer, block the membrane with 2 mL of 5% bovine serum albumin (BSA) in PBST (PBS with 0.1% Tween) overnight at 4 &#176;C following standard protocols11.</li>
</ol>
5. Antibody Labeling
<ol>
	<li>Incubate the membrane with antibodies on a shaker overnight at 4 &#176;C using antibody dilutions recommended by the vendor. 
	NOTE: In the Table of Materials, antibodies, which we have validated for HSCs and HPs and the corresponding antibody dilutions, are shown. Perform the antibody labeling using standard procedures8.</li>
</ol>
6. Detection
<ol>
	<li>Wash the membrane 3 times, 5 min for each wash in PBST at room temperature.</li>
	<li>Incubate the membrane with 2 mL of enhanced chemiluminescence buffer for 1 min at room temperature. Detect the signals using an imaging system following the manufacturer&#39;s instructions, or autoradiography film and film processor.</li>
</ol>

<ol>
	<li>Krutzik, P. O., Clutter, M. R., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+analysis+of+murine+immune+cell+surface+markers+and+intracellular+phosphoproteins+by+flow+cytometry.">Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.</a> J Immunol. 175 (4), 2357-2365 (2005).</li><li>Du, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+profiling+at+the+single-cell+level+identifies+a+distinct+signaling+signature+in+murine+hematopoietic+stem+cells.">Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells.</a> Stem Cells. 30 (7), 1447-1454 (2012).</li><li>Signer, R. A., Magee, J. A., Salic, A., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cells+require+a+highly+regulated+protein+synthesis+rate.">Haematopoietic stem cells require a highly regulated protein synthesis rate.</a> Nature. 509 (7498), 49-54 (2014).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differentiation+checkpoint+limits+hematopoietic+stem+cell+self-renewal+in+response+to+DNA+damage.">A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage.</a> Cell. 148 (5), 1001-1014 (2012).</li><li>Hoshii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+is+essential+for+leukemia+propagation+but+not+stem+cell+self-renewal.">mTORC1 is essential for leukemia propagation but not stem cell self-renewal.</a> J Clin Invest. 122 (6), 2114-2129 (2012).</li><li>Signer, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rate+of+protein+synthesis+in+hematopoietic+stem+cells+is+limited+partly+by+4E-BPs.">The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.</a> Genes Dev. 30 (15), 1698-1703 (2016).</li><li>Cai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx1+deficiency+decreases+ribosome+biogenesis+and+confers+stress+resistance+to+hematopoietic+stem+and+progenitor+cells.">Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells.</a> Cell Stem Cell. , (2015).</li><li>Loven, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+global+gene+expression+analysis.">Revisiting global gene expression analysis.</a> Cell. 151 (3), 476-482 (2012).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemoacytometer+to+Count+Cells.">Using a Hemoacytometer to Count Cells.</a> Basic Methods in Cellular and Molecular Biology. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+Protein+with+SDS-PAGE.">Separating Protein with SDS-PAGE.</a> Basic Methods in Cellular and Molecular Biology. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> Basic Methods in Cellular and Molecular Biology. , (2017).</li><li>Jackson, R. J., Hellen, C. U., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> Nat Rev Mol Cell Biol. 11 (2), 113-127 (2010).</li><li>Eaton, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> J Vis Exp. (93), e52099 (2014).</li></ol>

The authors have no conflict of interests to declare.

Western Blotting is a common technique for detecting specific proteins and the activation of signaling pathways in tissues or cells. By introducing small adjustments to a commonly used procedure, we were able to routinely detect 15 different proteins (Table of Materials) in 15,000 HSCs, and in some cases in as few as 500 HSCs. The most critical steps in this protocol are: 1) accurately counting the cells, 2) minimizing the number of transfers between tubes, and 3) lysing the cells directly with Laemmli s...

Hematopoietic stem cells (HSCs) are self-renewing cells that can give rise to all blood lineages. They are relatively rare cells in the bone marrow, rendering biochemical analyses difficult. Approaches suitable for analyzing rare cells, such as flow cytometry, have been extremely useful for quantifying relative amounts of cell surface markers and intracellular proteins. However, the analysis of intracellular proteins necessitates the use of cell permeabilization procedures to enable antibody access, and not all cell surface epitopes survive these procedures1,2. In addition, antibodies that discriminate between different protein isoforms or cleavage products are not often available for flow cytometry, and therefore investigators still rely on Western blots for certain types of analyses.
Western blot analysis of cell lysates is a routine procedure in most laboratories. Cells can be purified under native conditions that preserve the epitopes of cell surface molecules, and cell lysates can subsequently be prepared and analyzed. However, the analysis of proteins in rare primary cell populations by Western blot can require euthanizing large numbers of animals to obtain enough cells. By making small adjustments to several steps, a conventional Western blotting protocol was able to detect proteins in relatively small numbers of HSCs (500 - 15,000, depending on the protein of interest). The adjustments include accurately counting the cells, carefully handling the cell pellet, reducing transfers of cells between tubes to minimize cell loss, and lysing a defined number of cells with a concentrated loading buffer containing proteasome and phosphatase inhibitors. Many published reports include Western blots obtained with 20,000 or more HSCs3,4,5,6,7; this simple procedure will reduce the number of cells and experimental animals required to produce equivalent data by between 4 and 40 fold. The protocol is designed to normalize results on a per cell basis, rather than to an internal control. This enables detection of overall reductions in protein levels that can be overlooked if data are normalized to an internal control. The importance of normalizing on a per cell basis was described for the analysis of gene expression data8, and the same principle applies to quantifying proteins by Western blot. This optimized protocol should be useful for anyone needing to analyze small numbers of cells.

<table><tbody><tr><td>sodium dodecyl sulfate (SDS)</td><td>Fisher Scientific</td><td>BP166-500</td><td>SDS-PAGE</td></tr><tr><td>TEMED</td><td>Fisher Scientific</td><td>BP150-20</td><td>SDS-PAGE</td></tr><tr><td>30% Acrylamidel Bis solution</td><td>Bio-Rad</td><td>1610158</td><td>SDS-PAGE</td></tr><tr><td>1.5M Tris-HCl pH8.8</td><td>TEKNOVA</td><td>T1588</td><td>SDS-PAGE</td></tr><tr><td>1.0M Tris-HCl pH6.8</td><td>TEKNOVA</td><td>T1068</td><td>SDS-PAGE</td></tr><tr><td>Ammonium Persulfate</td><td>Fisher Scientific</td><td>BP179-25</td><td>SDS-PAGE</td></tr><tr><td>mini-protean 3 comb</td><td>Bio-Rad</td><td>1653360</td><td>SDS-PAGE</td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>1658005EDU</td><td>SDS-PAGE</td></tr><tr><td>Transfer System</td><td>Bio-Rad</td><td>1704155EDU</td><td>transfer</td></tr><tr><td>PowerPac HC Power Supply</td><td>Bio-Rad</td><td>1645052EDU</td><td>electrophoresis</td></tr><tr><td>Imaging System</td><td>Bio-Rad</td><td>1708195</td><td>signal detection</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747EDU</td><td>sample preparation</td></tr><tr><td>10x Tris/Glycine/SDS Electrophoresis Buffer</td><td>Bio-Rad</td><td>1610732EDU</td><td>electrophoresis</td></tr><tr><td>2-Mercaptoethanol</td><td>Bio-Rad</td><td>1610710EDU</td><td>sample preparation</td></tr><tr><td>RTA Mini PVDF Transfer Kit, for 40 blots</td><td>Bio-Rad</td><td>1704272</td><td>transfer</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>11697498001</td><td>sample preparation</td></tr><tr><td>Phosphatase Inhibitor Cocktailrs</td><td>Gold Biotechnology</td><td>GB-450-1</td><td>sample preparation</td></tr><tr><td>EIF4G</td><td>Cell signaling</td><td>2469</td><td>WB antibody, validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>p-Rps6</td><td>Cell signaling</td><td>4858</td><td>WB antibody,validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>actin(HRP conjugated)</td><td>abcam</td><td>ab49900</td><td>WB antibody,validated in 500 cells&lt;br/&gt; antibody concentration: 1:5000&lt;br/&gt; reference(figure): Fig1, Fig2</td></tr><tr><td>LC-3</td><td>Abcam</td><td>ab48394</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>S6</td><td>Cell Signaling</td><td>2317</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>4EBP1</td><td>Cell Signaling</td><td>9644</td><td>WB antibody, validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>p-4EBP1</td><td>Cell Signaling</td><td>2855</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>TSC2</td><td>Cell Signaling</td><td>4308</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>HSP90</td><td>Cell Signaling</td><td>4877</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>PTEN</td><td>Cell Signaling</td><td>9188</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>mTOR</td><td>Cell Signaling</td><td>2983</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-mTOR</td><td>Cell Signaling</td><td>5536</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>PP2AB</td><td>Cell Signaling</td><td>4953</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-AMPKa</td><td>Cell Signaling</td><td>2535</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>actin</td><td>Santa Cruz</td><td>sc-47778</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:3000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>lineage depletion kit (mouse)</td><td>Miltenyi Biotec</td><td>130-090-858</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-041-305</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>SCF</td><td>PeproTech</td><td>250-03</td><td>phosphorylation stimulation</td></tr><tr><td>APC-Cy7 c-kit antibody</td><td>ThermoFisher</td><td>A15423</td><td>cell sorting</td></tr><tr><td>anti-B220</td><td>ThermoFisher</td><td>48-0452-82</td><td>cell sorting</td></tr><tr><td>anti-CD3e</td><td>ThermoFisher</td><td>48-0031-82</td><td>cell sorting</td></tr><tr><td>anti-Mac1</td><td>ThermoFisher</td><td>48-0112-82</td><td>cell sorting</td></tr><tr><td>anti-Gr1</td><td>ThermoFisher</td><td>48-5931-82</td><td>cell sorting</td></tr><tr><td>anti-Ter119</td><td>ThermoFisher</td><td>48-5921-82</td><td>cell sorting</td></tr><tr><td>Spacer Plates with 1.0 mm Integrated Spacers</td><td>Bio-Rad</td><td>1653311</td><td>SDS-PAGE</td></tr><tr><td>BSA</td><td>Research Products International</td><td>A30075</td><td>membrane blocking</td></tr><tr><td>serum</td><td>Atlanta Biologicals</td><td>A12450</td><td>medium supplement</td></tr><tr><td>cell counter</td><td>Invitrogen</td><td>AMQAF1000</td><td>cell counting</td></tr><tr><td>hemocytometer</td><td>ThermoFisher</td><td>&amp;nbsp;S17040</td><td>cell counting</td></tr><tr><td>flim</td><td>Denville Scientific</td><td>E3012</td><td>signal detection</td></tr><tr><td>medical film processor</td><td>Konica</td><td>SRC-101A</td><td>signal detection</td></tr><tr><td>Supersignal West Femto Maximum Sensitivity Substrate</td><td>Therom Scientific</td><td>34096</td><td>signal detection</td></tr><tr><td>Allegra X-15R Centrifuge (Rotor SX4750A)</td><td>Beckman Coulter</td><td>X-15R</td><td>sample preparation</td></tr><tr><td>BLUEstain protein ladder</td><td>Gold Biotechnology</td><td>P007-500</td><td>electrophoresis</td></tr><tr><td>cell sorter</td><td>BD</td><td>FACSAria II</td><td>sample preparation</td></tr><tr><td>Incublock</td><td>Denville Scientific</td><td>I0510</td><td>electrophoresis</td></tr></tbody></table>

a western blotting protocol for small numbers of hematopoietic stem cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

Representative results from 500 - 2,000 purified HSCs and HPs are shown in Figure 1 and Figure 2. The &#946;-actin signal in Figure 1 can be detected from as few as 500 HSCs and HPs purified from the bone marrow of one mouse. Note that loading the lysates into 1.5 mm wells produced a much stronger signal from 500 HPs than loading into 3.0 mm wells. Figure 2 is a Western ...

Watch this Scientific Journal Video about A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells at JoVE.com

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

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10.3K Views.  Cincinnati Children's Hospital Medical Center. Een standaard Western blotting protocol is geoptimaliseerd voor het analyseren van slechts 500 hematopoietische stamcellen of voorlopercellen. Optimalisatie houdt zorgvuldige behandeling van het monster van de cel, beperking van de overdracht tussen buizen en direct het lysing van cellen in Laemmli monster buffer.

Video: A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Biology

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Developmental Biology

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Bioengineering

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

Immunology and Infection

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),&#160;defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

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