We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St Louis, MO, for the use of the High Speed Cell Sorter Core, which provided flow cytometry equipment and sorting services.  This work was supported by National Institutes of Health Grants R01 HL082549 and K02 HL084179 (NRY).  The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant P30 CA91842. 

1. PREPARATION OF REAGENTS
<ol>
<li> Prepare sterile 1000x stock solutions of Fms-related tyrosine kinase 3 (FLT-3) ligand, granulocyte/macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), thrombopoietin (TPO), interleukin (IL)-3, and IL-6 according to manufacturer's instructions. Prepare sterile Retronectin stock solution (1mg/ml) also according to manufacturer's instructions. Divide these stock solutions into small aliquots and keep at -20 &deg;C to avoid repeated freeze-thaw.</li>
<li> Prepare 2% FBS in IMDM.</li>
<li> Prepare complete IMDM medium with final concentrations of 20% FBS, 2 mM Glutamine, 100 units/ml Penicillin/Streptomycin (PS). Add the following cytokines just before use: 100 ng/ml FLT-3 ligand, 20 ng/ml GM-CSF, 100 ng/ml SCF, 100 ng/ml TPO, 50 ng/ml IL-3, and 100 ng/ml IL-6.</li>
<li> Prepare 1% BSA in HBSS (Ca &amp; Mg-free, without phenol red) and 2% BSA in D-PBS (Ca &amp; Mg-free). Sterilize the solutions by passing through 0.22 &mu;m syringe filters and store at 4 &deg;C.</li>
<li> Prepare 0.02% EDTA in HBSS and store at 4&deg;C.</li>
<li> Prepare GaLV-pseudotyped retrovirus encoding the DNA of interest and store in aliquots at -80 &deg;C. This procedure is outside the scope of this article, but is described elsewhere (1-3).</li>
</ol>
For Giemsa Staining
<ol start="7">
<li> Just before use, filter the Wright/Giemsa stain solution twice through 1 &mu;m filter papers into a clean glass bottle.</li>
<li> Immediately before use, prepare Wright/Giemsa buffer pH 6.4, by mixing 50 ml of the filtered Wright/Giemsa stain solution and 200 ml of phosphate buffer solution, pH 6.4 - Giordano formula.</li>
<li> Prepare phosphate buffer solution, pH 6.0 by dissolving 27.3 g of KH2PO4 and 4.62 g of Na2HPO4 in 3.5 L H2O.</li>
<li> Prepare 50% methanol in H2O.</li>
</ol>

2. PROCEDURE
Thawing and pre-activation of CD34+ cells
<ol>
<li> Thaw a vial of frozen CD34+ cells quickly at 37 &deg;C by gently shaking until a last small ice crystal is left and transfer the cell suspension (1 ml) to a 50 ml conical tube. Gently rinse off the remaining cells from the vial with 1 ml of room temperature 2% FBS/IMDM and add it drop-wise to the 50 ml tube while swirling gently. Wait for 3 minutes. Next, slowly add 2 ml of 2% FBS/IMDM, while mixing gently, and wait for 3 minutes. Repeat the procedure by adding 2% FBS/IMDM that is the same volume as the diluted cell suspension at 3 min intervals, swirling gently in between additions, until the final volume reaches 32 ml.</li>
<li> Centrifuge at 250 x g for 10 minutes at room temperature and remove the supernatant leaving behind around 0.5 ml.</li>
<li> Wash the pellet by suspending in 20 ml 2% FBS/IMDM and centrifuging at 200 x g for 8 minutes at room temperature.</li>
<li> Remove the supernatant and suspend the cells in complete IMDM medium at approximately 0.5 x 106 cells/ml. Take 10 &mu;l of the cell suspension into a microtube, mix with the same volume of Trypan blue solution (0.4%) and count using a hemocytometer.</li>
<li> Dilute the cells to 0.1-0.15 X 106 cells/ml by adding complete IMDM medium supplemented with cytokines (see 1.3)</li>
<li> Culture the cells in a humidified 37&deg;C incubator with 5% CO2 for 2 days.</li>
</ol>
Retroviral transduction by virus pre-loading
<ol start="7">
<li> On the day of transduction, count cells before starting virus preloading to determine the number of wells to be prepared.</li>
<li> Dilute the Retronectin stock solution to 25 &mu;g/ml and add 400 &mu;l to each well of a 24-well non-treated tissue culture plate. Incubate for 2 hours at room temperature in the laminar-flow biosafety hood to pre-coat.</li>
<li> Remove the Retronectin solution and add 400 &mu;l of 2% BSA in D-PBS to each well. Incubate for 30 minutes at room temperature.</li>
<li> Thaw virus stock solutions and keep on ice. Remove the BSA solution, add 0.5 ml of virus preparation to each well, and centrifuge at 2200 x g at 4&deg;C for 15 minutes.</li>
<li> Remove the virus solution from wells and repeat the virus loading described in 2.10 three more times.</li>
<li> Rinse each well with cold 0.5 ml HBSS (+ Ca &amp; Mg) or IMDM.</li>
<li> Collect the pre-activated CD34+ cells by centrifuging at 200 x g for 8 minutes at room temperature.</li>
<li> Resuspend the cells at 0.1-0.15 x 106 cells per 1.5 ml in complete IMDM medium with cytokines.</li>
<li> Add 1.5 ml cell suspension to each well and incubate in a humidified 37&deg;C incubator with 5% CO2 for 2 days.</li>
</ol>
Cell sorting and collection of transduced cells
<ol start="16">
<li> Gently but thoroughly suspend the cells in the above virus-loaded plates and filter through a 50 &mu;m CellTrics cell filter into a conical tube. Take 10 &mu;l of the cell suspension into a microtube, mix with an equal volume of Trypan blue solution and count using a hemocytometer. Rinse each well with 0.8 ml of cold 0.02% EDTA/HBSS (Ca &amp; Mg-free without phenol red) and add to the same collection tube through a 50 &mu;m CellTrics cell filter.</li>
<li> Centrifuge the cells at 200 x g for 8 minutes at 4&deg;C, and wash the pellet with cold HBSS (Ca &amp; Mg-free without phenol red) by centrifuging at 200 x g for 8 minutes at 4&deg;C.</li>
<li> Resuspend the pellet in 1% BSA/HBSS (Ca &amp; Mg-free without phenol red) to a concentration of 15 X 106/ml or a minimum volume of 0.250 ml for cell sorting. Keep the cells on ice in the dark. Prepare a tube containing cold 20% FBS/IMDM/Glutamine/PS medium for collecting the sorted cells. Sorting is performed at the High Speed Cell Sorter Core of the Alvin J. Siteman Cancer Center at Washington University School of Medicine using a MoFlo high-speed sorter (Dako, Glostrup, Denmark).</li>
</ol>
CFC assay
<ol start="19">
<li> Thaw the required number of 3 ml aliquots of CFC assay media. Vortex vigorously to mix and let the tubes stand at least 5 min to let any bubbles present to rise to the surface before adding cells.</li>
<li> To obtain the cell concentration for each sorted sample, divide the cell number provided by the sorting facility by the approximate cell suspension volume. Take 3,000 virus-transduced, sorted cells into a sterile microtube containing cold 2% FBS/IMDM. Pre-adjust the volume of 2% FBS/IMDM so that the final suspension volume will be approximately 0.3 ml.</li>
<li> Suspend the cells and transfer the entire 0.3 ml cell suspension to a 3 ml aliquot of Methocult GF+ H4435, which consists of 1% methylcellulose, 30% FBS, 1% BSA, 10-4 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-3, 20 ng/ml IL-6, 20 ng/ml G- CSF, and 3 units/ml erythropoietin in IMDM. Human Methylcellulose Enriched Media (R &amp; D Systems) which consists of 1.3% methylcellulose, 25% FBS, 1% BSA, 5 x 10-5 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-3, 20 ng/ml IL-6, 20 ng/ml G- CSF, and 3 units/ml erythropoietin in IMDM can be also used.</li>
<li> Vortex vigorously to make the mixture rise fully and fall 3-4 times. Let the tube stand still for 3 min.</li>
<li> Attach a 16 gauge blunt-end needle to a 3 ml syringe and draw up 2.2 ml. Do not draw up large bubbles; expel them at the beginning by pushing out a couple of times. Push out 1.1 ml each into two 30 mm non-treated dish and spread out the mixture evenly by rotating.</li>
<li> Place duplicate plates in a 100 mm plate together with a water dish containing 3 ml sterile water. Culture for 14 - 17 days.</li>
<li> Characterize and score the colonies according to their morphology with an inverted microscope at 40x magnification in a culture dish marked with a scoring grid. For the purposes of our assay, the colonies are classified into 3 categories: pure erythroid, myelomonocytic, and mixed. See manufacturer's instructions for further colony subclassification.</li>
<li> The entire CFC assay plate may be scanned without magnification using a regular scanner at 600 dpi, and low-power (40x) photomicrographs of colonies may be taken using an inverted microscope equipped with a color camera.</li>
<li> For further analysis of differentiation and proliferation, cells from the entire CFC assay plate are recovered by suspending in several volumes of room temperature 2% FBS/IMDM. After centrifugation at 300 x g for 10 min at 4&deg;C, the cells are resuspended in 2% FBS/IMDM, counted, and either stained with antibodies for flow cytometry or transferred to slides using a Cytospin centrifuge for Giemsa staining.</li>
</ol>
Antibody staining and flow cytometry
<ol start="28">
<li> Centrifuge cells at 200 x g for 8 min at 4&deg;C and resuspend in cold 50% normal mouse serum in HBSS (Ca &amp; Mg-free without phenol red).</li>
<li> Place approximately 5 x 105 cells in 80 &mu;l per tube (12 x 75 mm round-bottom tubes) and keep on ice. Add antibody mixture (approximately 20 &mu;l mixture or negative control solution for no-antibody controls) and mix by lightly tapping the tubes. Prepare calibration control tubes with no antibody or with an anti-CD45 antibody for each fluorochrome. Place tubes on ice in the dark and incubate for 20 minutes. Mix by lightly tapping tubes after the first 10 min. After the 20 min incubation, fill the tubes with cold HBSS and centrifuge at 200 x g for 8 min at 4&deg;C. Resuspend the pellet in cold 0.5% paraformaldehyde/HBSS. 
The following mixtures of antibodies are typically used for our analysis. For myeloid differentiation: CD11b (phycoerythrin-conjugated clone ICRF44)/CD33 (allophycocyanin-conjugated clone WM53)/CD45 (phycoerythrin-Cy7-congjugated clone HI30). For erythroid differentiation: CD71 (phycoerythrin-conjugated clone M-A712)/CD235a (allophycocyanin-conjugated clone GA-R2)/CD45 (phycoerythrin-Cy7-congjugated clone HI30).</li>
<li> Flow cytometry is performed at the High Speed Cell Sorter Core of the Alvin J. Siteman Cancer Center at Washington University School of Medicine on a FACScan flow cytometer upgraded to 5 colors and two lasers (BD Biosciences) and analyzed using CellQuest (BD Biosciences) or FlowJO v7.2.4 (Tree Star, Inc., Ashland, OR, USA) software.</li>
</ol>
Giemsa staining
<ol start="31">
<li> Approximately 30,000 cells recovered from CFC assay plates described above are suspended in 0.3 ml 2% FBS/IMDM and transferred to a slide using a Cytospin centrifuge at 1,000 rpm for 10 min at room temperature.</li>
<li> Set up nine staining vessels containing solutions in the following order.
<ol>
<li> Absolute Methanol</li>
<li> Wright/Giemsa stock solution</li>
<li> Wright/Giemsa buffer, pH 6.4</li>
<li> 50% Methanol in H2O</li>
<li> H2O</li>
<li> H2O</li>
<li> Phosphate buffer, pH 6.0</li>
<li> H2O</li>
<li> H2O</li>
</ol></li>
<li> Place the slides into a slide carrier and dip in absolute methanol (#1) for 2 min and blot off excess methanol.</li>
<li> Immediately, dip the slide carrier in Wright/Giemsa stock solution (#2) for 5 min.</li>
<li> Transfer the carrier into Wright/Giemsa buffer, pH 6.4 (#3) and incubate for 10 min.</li>
<li> Dip the carrier twice in 50% methanol (#4), 10 times in H2O (#5), another 10 times in H2O (#6), and then 5 times in Phosphate buffer, pH 6.0 (#7).</li>
<li> Transfer the carrier into H2O (#8) and incubate for 2 min. Repeat the wash in H2O (#9).</li>
<li> Allow the slides to air dry completely in the carrier. Remove each slide and wipe the back of the slide with methanol-soaked Kimwipes to remove stains. Place a drop of Cytoseal 60 and a cover glass to seal.</li>
<li> A 500-cell differential count is performed for each Giemsa-stained slide using an Olympus BX51 microscope. Cells are divided into five categories: primitive cells include blasts and promyelocytes; intermediate myeloid cells include myelocytes and metamyelocytes; mature myeloid cells include bands, segmented neutrophils, monocytes, and macrophages; intermediate erythroid cells include cells with intermediate hemoglobinization; and mature erythroid cells include cells with full hemoglobinization. Photomicrographs are taken with an Olympus DP71 camera with a 60x oil objective.</li>
</ol>

3.0) REPRESENTATIVE RESULTS:
<ol>
<li>CFC assay: Some of the input cell population will proliferate, differentiate, and form colonies on the semi-solid medium during the 14-17-day incubation period. In this experiment, it is evident that expression of the NUP98-HOXA9 oncogene caused the formation of prominent red (erythroid colonies) (Figure 1) (3).</li>
<li>Antibody Staining and Flow Cytometry: Flow cytometric immunophenotyping of cells collected from CFC assay plates provides information on the differentiation state of the cell population. By combined use of antibodies against certain differentiation markers, the lineage and degree of maturation of cells can be assessed. In this case CD235a was used to identify erythroid cells. As shown in Figure 2A, NUP98-HOXA9 caused an increase in the percentage of CD235a+ erythroid cells, but the brightness of CD235a expression by these cells was diminished compared to controls, indicating inhibition of erythroid maturation. In Figure 2B, myeloid cells are identified by virtue of their CD33 positivity. NUP98-HOXA9 caused a decrease in the numbers of CD33+ cells, with a decrease in the brightness of CD11b expression, consistent with inhibition of myeloid maturation (3). Thus the flow cytometry results show that NUP98-HOXA9 caused an erythroid hyperplasia with inhibition of both myeloid and erythroid maturation.</li>
<li>Giemsa Staining: Morphological examination of cells collected from CFC assay plates provides information on the lineage and degree of maturation of the cell population. The conclusions obtained morphologically may differ from those obtained by flow cytometric studies, and thus these methods are complementary to each other (3). The experiment is repeated at least 3 times, and each time a 500-cell differential count is performed to distinguish 5 categories of cells as described above. Examples of these cells are pointed out in Figure 3. The results are tabulated and analyzed in order to determine whether there are statistically significant differences between the oncogene-transduced sample and the control. In this case, the results showed that NUP98-HOXA9 caused an overall increase in the numbers of cells, with erythroid hyperplasia and inhibition of both erythroid and myeloid maturation (3) (not shown).</li>
</ol>
FIGURE LEGENDS
<img src="/files/ftp_upload/2195/2195fig1.jpg" alt="Figure 1" /> 
Figure 1: The effects of NUP98-HOXA9 on human CFC morphology. 
Primary human CD34+ cells were retrovirally transduced with either control MSCV-IRES-GFP vector or vector expressing NUP98-HOXA9, and cells were sorted for GFP positivity. One thousand cells were seeded into each of two duplicate plates for CFC assay and the experiment was repeated 3 4 independent times. Representative plates without magnification (left) and low power photomicrographs of representative erythroid colonies (right) are shown (3).

<img src="/files/ftp_upload/2195/2195fig2.jpg" alt="Figure 2" /> 
Figure 2: Flow cytometry shows disruption of human primary CD34+ cell differentiation by NUP98-HOXA9. 
(A) Flow cytometry for erythroid differentiation: Cells from the CFC plates (see Figure 1) were harvested and stained with antibodies to CD45 and CD235a. The CD235a+ gate was plotted on a histogram (right panel) to show the expression of CD235a relative to control cells (B) Flow cytometry for myeloid differentiation: Cells from the CFC plates were harvested and stained with CD45 and CD33. The CD33+ gate was plotted on a histogram to show CD11b expression compared to control (right panel). The percentages of cells falling within each gate are shown (3).
<img src="/files/ftp_upload/2195/2195fig3.jpg" alt="Figure 3" /> 
Figure 3: Cell morphology shows disruption of differentiation by NUP98-HOXA9 compared to vector control. 
Cytospin smears were prepared from CFC plates and stained with Giemsa. Photomicrographs were taken from representative fields with a 60x oil objective. B: blast; MM: mature myeloid, IM: intermediate myeloid, ME: mature erythroid, IE: intermediate erythroid (3).

<ol>
	<li>Takeda, A., Goolsby, C., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98-HOXA9+induces+long-term+proliferation+and+blocks+differentiation+of+primary+human+CD34++hematopoietic+cells.">NUP98-HOXA9 induces long-term proliferation and blocks differentiation of primary human CD34+ hematopoietic cells.</a> Cancer Res. , 66-6628 (2006).</li><li>Yassin, E. R., Abdul-Nabi, A. M., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+NUP98-DDX10+oncogene+on+primary+human+CD34++cells:+role+of+a+conserved+helicase+motif.">Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: role of a conserved helicase motif.</a> Leukemia. , (2010).</li><li>Yassin, E. R., Sarma, N. J., Abdul-Nabi, A. M., Dombrowski, J., Han, Y., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+transformation+of+primary+human+hematopoietic+cells+by+the+oncogene+NUP98-HOXA9.">Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.</a> PLoS One. 4, e6719-e6719 (2009).</li><li>Nissen-Druey, C., Tichelli, A., Meyer-Monard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hematopoietic+colonies+in+health+and+disease.">Human hematopoietic colonies in health and disease.</a> Acta Haematol. 113, 5-96 (2005).</li><li>Pereira, C., Clarke, E., Damen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cell+assays.">Hematopoietic colony-forming cell assays.</a> Methods Mol Biol. 407, 177-208 (2007).</li><li>Coulombel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hematopoietic+stem/progenitor+cells:+strength+and+drawbacks+of+functional+assays.">Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.</a> Oncogene. 23, 7210-7222 (2004).</li><li>Hogge, D. E., Lansdorp, P. M., Reid, D., Gerhard, B., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection,+maintenance,+and+differentiation+of+primitive+human+hematopoietic+cells+in+cultures+containing+murine+fibroblasts+engineered+to+produce+human+steel+factor,+interleukin-3,+and+granulocyte+colony-stimulating+factor.">Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor.</a> Blood. 88, 3765-3773 (1996).</li></ol>

No conflicts of interest declared.

The CFC assay has been used extensively to determine the proliferation and differentiation patterns of hematopoietic progenitors and to study the effects of oncogenes (4, 5). It has the advantage over liquid cultures of being a clonal assay, such that the colonies represent the progeny of a single progenitor and can be individually removed for further analysis. The limitation of the CFC assay is that it is not adequate for the detection of more immature progenitors or hematopoietic stem cells; such cells are detected u...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>12440</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>06150</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin (PS)</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>FLT-3 ligand</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-19</td>
<td>&nbsp;</td>
<tr>
<td>GM-CSF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-03</td>
<td>&nbsp;</td>
<tr>
<td>SCF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-07</td>
<td>&nbsp;</td>
<tr>
<td>TPO</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-18</td>
<td>&nbsp;</td>
<tr>
<td>IL3</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-03</td>
<td>&nbsp;</td>
<tr>
<td>IL6</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-06</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP118</td>
<td>&nbsp;</td>
<tr>
<td>Retronectin</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>T100A</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14200</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue solution (0.4%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
<tr>
<td>Methocult GF+ H4435</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>04445</td>
<td>&nbsp;</td>
<tr>
<td>Human Methylcellulose Enriched Media</td>
<td>&nbsp;</td>
<td>R &amp; D Systems</td>
<td>HSC005</td>
<td>&nbsp;</td>
<tr>
<td>Wright/ Giemsa stain</td>
<td>&nbsp;</td>
<td>Harleco</td>
<td>64571</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Solution, pH 6.4 - Giordano formula</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>1450</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A412-4</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 60</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>8310</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum </td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals</td>
<td>D208</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD11b phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555388</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD33 allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551378</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD71 phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555537</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD235a allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551336</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>24-well non-treated tissue culture plates</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-1147</td>
<td>&nbsp;</td>
<tr>
<td>30 mm non-treated dish</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27150 </td>
<td>&nbsp;</td>
<tr>
<td>100 mm tissue culture dish </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27500</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2097</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2070</td>
<td>&nbsp;</td>
<tr>
<td>Syringes 3 ml </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28240</td>
<td>&nbsp;</td>
<tr>
<td>16 gauge blunt-end, 1&frac12; inch needle </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28110</td>
<td>&nbsp;</td>
<tr>
<td>50 &mu;m CellTrics cell filter</td>
<td>&nbsp;</td>
<td>Partec</td>
<td>04-004-2327</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0267110</td>
<td>&nbsp;</td>
<tr>
<td>TPX sample chambers</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>A78710018</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Superfrost/Plus Microscope Slides, Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>Shandon filter cards</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>5991022</td>
<td>&nbsp;</td>
<tr>
<td>Shandon cytospin slide holder</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>59920063</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Complete Staining Assembly 100</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>100</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark Corporation</td>
<td>34155</td>
<td>&nbsp;</td>
<tr>
<td>1 &mu;m filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28307-134</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Diaphot</td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>DS-F11</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX51 </td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DP71</td>
<td>&nbsp;</td>
<tr>
<td>Scanner</td>
<td>&nbsp;</td>
<td>Microtek</td>
<td>Scanmaker 4</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-812</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator</td>
<td>&nbsp;</td>
<td>Sanyo</td>
<td>MCO-18AIC</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>&nbsp;</td>
<td>Shandon</td>
<td>Cytospin 2</td>
<td>&nbsp;</td>
<tr>
<td>Bench-top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5810-R</td>
<td>&nbsp;</td>
<tr>
<td>Water purification system</td>
<td>&nbsp;</td>
<td>Barnstead</td>
<td>Nanopure-Diamond</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

colony forming cell (cfc) assay for human hematopoietic cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Watch this Scientific Journal Video about Colony Forming Cell CFC Assay for Human Hematopoietic Cells at JoVE.com

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

colony-forming-cell-cfc-assay-for-human-hematopoietic-cells

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JoVE Journal

Immunology and Infection

35.5K Views.  Washington University School of Medicine. The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Video: Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...