This study was supported by grants from the Swiss National Science Foundation (179239), the Foundation for Fight Against Cancer (Zuerich),&#160;the Wilhelm Sander Foundation to CL (2019.042.1), and the Novartis Foundation for medical-biological research to C.L. Furthermore, this project has received funding from the European Union&#8217;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement No. 765104. We thank the Flow Cytometry Facility in Basel for support.

Patient samples were collected following approval from the Ethics Review Board of the University Hospitals of Basel and Tuebingen.
1. Biotinylation of the NKG2D fusion protein
NOTE: This step is performed with a biotinylation kit (see Table of Materials) according to the manufacturer&#8217;s instruction. This step of the protocol must be performed at least 24 h prior to the staining. The biotinylated NKG2D fusion protein should be stored at -20 &#176;C.
<ol>
	<li>Thaw both the biotin, as well as the NKG2D fusion protein tubes at room temperature (RT). 
	NOTE: If the cells need to be sterile for further experimental use (injection in mice, colony forming unit assays, etc.), reconstitute the fusion protein under a laminar flow hood to avoid contamination. Otherwise, each step can be performed on a bench.</li>
	<li>Quickly spin down the 50&#181;g of lyophilized NKG2D fusion protein. Add 500 &#181;L of phosphate-buffered saline (PBS) to reconstitute the powder and mix thoroughly using a P1000 micropipette.</li>
	<li>Add 100 &#181;L of the NKG2DL fusion protein to a biotin tube to obtain a final stock concentration of 100 &#181;g/mL.</li>
	<li>Mix the solution thoroughly by continuous resuspending with a P100 micropipette.</li>
	<li>Incubate the fusion protein/biotin solution at a controlled RT for 24 h. The fusion protein is now ready to use.</li>
</ol>
2. Thawing of primary AML cells
NOTE: Approximately 5,000,000 frozen leukemic cells per patient stored in liquid nitrogen were thawed and then used for the assays right away. Leukemic cells were obtained and frozen as previously described15. Briefly, peripheral blood samples collected from patients with AML and high blast cell percentages (&#62;90% of blasts among mononuclear cells) were processed with a density gradient separation to obtain mononuclear cells and subsequently frozen in fetal calf serum (FCS) containing 10% dimethyl sulfoxide solution (DMSO). Cell numbers highly varied between patients in dependence on the leukocyte concentration in the patient (range: 1,000,000 to 30,000,000 leukemic cells per mL blood).
<ol>
	<li>Pre-warm RPMI medium containing 10% of FCS at 37 &#176;C using a water bath. For each vial of AML cells (up to 30,000,000 cells in 1 mL FCS + 10% DMSO), add 10 mL of pre-warmed medium to a 15 mL reaction tube.</li>
	<li>Remove the cryovial containing primary AML from the liquid nitrogen storage and immediately thaw the cells using a 37 &#176;C water bath. Gently move the tube back and forth in the water, allowing the contents of the vial to thaw until there is only a small ice crystal left.</li>
	<li>Immediately transfer the thawed cells into the medium-containing tube and rinse the vial using 1 mL of medium.</li>
	<li>Centrifuge the cells at 300 x g for 10 min and discard the supernatant without disturbing the pellet.</li>
	<li>Wash the cells with 5 mL of RPMI medium containing 10% of FCS and centrifuge at 300 x g for 10 min.</li>
</ol>
3. Cell counting
<ol>
	<li>Resuspend the cell pellet in a known volume of RPMI medium containing 10% of FCS.</li>
	<li>Transfer a small volume of the cell suspension to a 1.5 mL microcentrifuge tube and dilute at a known ratio with trypan blue or any other alternative dye that allows live cell/dead cell discrimination.</li>
	<li>Use any device to count the cells.</li>
	<li>Centrifuge the cells at 300 x g for 10 min and discard the supernatant without disturbing the pellet.</li>
</ol>
4. Staining of primary AML cells using the biotinylated NKG2D fusion protein
<ol>
	<li>Prepare the staining buffer by supplementing 500 mL PBS with bovine serum albumin (BSA) and Ethylenediaminetetraacetic acid (EDTA) to a final concentration of 0.07 mM and 2 mM, respectively. Adjust the pH (7.2&#8211;7.4) if necessary.</li>
	<li>Resuspend the cell pellet with staining buffer to a final concentration of 0.5 x 107 cells/mL. 
	NOTE: Optionally, prior to staining, the cells can be incubated with a blocking agent (e.g. hIgG (1&#181;g/ml) for 30 min at RT or FcR-blocking agent)..</li>
	<li>Transfer 100 &#181;L of the cell suspension to a cell culture 96-well U-bottom plate and centrifuge the plate at 300 x g for 10 min. Discard the supernatant without disturbing the pellet.</li>
	<li>Prepare a master mix of biotinylated NKG2D fusion protein so that cells are resuspended in a final volume of 50 &#181;L per well with a final concentration of 10 &#181;g/mL per well.</li>
	<li>Add the master mix prepared in step 4.4 using a 100 &#181;L pipette and resuspend the cell pellets with a 300 &#181;L multichannel pipette. 
	NOTE: Scale down the final volume according to the number of cells to reduce the amount of fusion protein necessary for the staining.</li>
	<li>Incubate the cell suspension for 25 min at RT or 50 min at 4 &#176;C.</li>
	<li>Wash the cells by adding 200 &#181;L of staining buffer per well with a 300 &#181;L multichannel pipette.</li>
	<li>Centrifuge the plate at 300 x g for 10 min and discard the supernatant without disturbing the pellet.</li>
	<li>Repeat steps 4.7 and 4.8. 
	NOTE: The staining described here is performed in a cell culture 96-well U-bottom plate. Therefore, washing steps are performed twice because of the small volume capacity of such plates. The staining can also be performed in other tubes and washing steps might then be performed only once using a larger volume.</li>
	<li>Prepare a master mix using Streptavidin-PE (see Table 1 for dilutions) so that cells are resuspended in a final volume of 50 &#181;L. 
	NOTE: Add selection antibodies for your cell type of interest as e.g. CD33, CD34&#8230;. for AML.</li>
	<li>Add the master mix prepared in step 4.10 using a 100 &#181;L pipette and resuspend the cell pellets with a 300 &#181;L multichannel pipette.</li>
	<li>Incubate the cell suspensions for 15 min at RT or 30 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells by adding 200 &#181;L of staining buffer per well with a 300 &#181;L multichannel pipette.</li>
	<li>Centrifuge the plate at 300 x g for 10 min and discard the supernatant without disturbing the pellet.</li>
	<li>Repeat steps 4.13 and 4.14.</li>
	<li>Prepare a solution of staining buffer including 7-AAD (7-Aminoactinomycin D, 1:1000) or any reagent to distinguish between live and dead cells.</li>
	<li>Resuspend cell pellets in 200 &#181;L staining buffer + 7-AAD prepared in step 4.16 in using a 300 &#181;L multichannel micropipette.</li>
	<li>Analyze the cells using a flow cytometry device.</li>
</ol>
5. Staining of single or pooled single anti-NKG2DL antibodies
<ol>
	<li>Use the same cell suspension as in step 4.2.</li>
	<li>Transfer 100 &#181;L of the cell suspension to a cell culture 96-well U-bottom plate using a 300 &#181;L multichannel pipette and centrifuge the plate at 300 x g for 10 min. Discard the supernatant without disturbing the pellet.</li>
	<li>Prepare an antibody master mix for each primary antibody or pooled antibodies (see Table 2 for dilutions) so that the cells are resuspended in a final volume of 50 &#181;L.</li>
	<li>Add the master mix prepared in step 5.3 using a 100 &#181;L pipette and resuspend the cell pellets with a 300 &#181;L multichannel pipette.</li>
	<li>Incubate the cells for 25 min at RT.</li>
	<li>Wash the cells by adding 200 &#181;L of staining buffer per well using a 300 &#181;L multichannel pipette.</li>
	<li>Centrifuge the cell culture 96-well U-bottom plate at 300 x g for 10 min and discard the supernatant without disturbing the pellet.</li>
	<li>Repeat steps 5.6 and 5.7.</li>
	<li>Prepare an antibody master mix by adding the secondary antibody (see Table 2 for dilutions) so that the cells are resuspended in a final volume of 50 &#181;L per well.</li>
	<li>Add the master mix prepared in step 5.9 using a 100 &#181;L pipette and resuspend the cell pellets with a 300 &#181;L multichannel pipette.</li>
	<li>Incubate for 15 min at RT or 30 min at 4 &#176;C in the dark.</li>
	<li>Wash by adding 200 &#181;L of staining buffer per well using a 300 &#181;L multichannel pipette.</li>
	<li>Centrifuge the cell suspension at 300 x g for 10 min and discard the supernatant without disturbing the pellet.</li>
	<li>Repeat steps 5.12 and 5.13.</li>
	<li>Resuspend cell pellets in 200 &#181;L staining buffer containing 7-AAD prepared in step 4.16.</li>
	<li>Analyze the cells using a flow cytometry device as described in step 6.</li>
</ol>
6. Data acquisition
NOTE: Make sure that weekly quality controls of the flow cytometer are performed to ensure that lasers are functioning properly. For the protocol presented here, a weekly Cytometer and Tracking (CTS) process is performed with relative beads to check laser performances on all channels. After the CTS, 10,000 events are recorded using the 8-peak beads as an internal control. All peaks should be in the same position inside their gates and well separated from each other.
<ol>
	<li>Create the matrix compensation to subtract spectral overlap between detectors with beads or with cells16.</li>
	<li>Record 10,000 events for the unstained cells and the single stained beads. For the fluorescence minus one (FMO) controls, record 50,000 events and for the full-stained samples record up to 100,000 events. 
	NOTE: Alternatively, single stained samples can be performed on the cells. If so, make sure that the cells have a positive signal for the desired marker.</li>
	<li>Acquire a single stained cell or bead sample for each fluorophore used in the experiment to reveal the amount of spectral overlap. Use the compensation creator of the flow cytometry device to calculate spill-over values and apply the compensation matrix to all the measured samples. 
	NOTE: Secondary antibodies cannot be used for compensation creation using beads if the compensation is calculated with beads. Depending on the type of compensation beads, secondary antibodies cannot be used for the compensation with beads.</li>
	<li>For the FMO controls and the full stained samples with the fusion protein, firstly, select the cells based on their forward scatter (FSC) and side scatter (SSC). After the exclusion of doublets, gate the cells based on their negativity for 7-AAD (PercP-Cy5.5) signal to exclude the dead cells (see step 5.15). The final plot shows the expression of CD34 (Y-axis) and NKG2DL (X-axis) on living singlets.</li>
	<li>For the single anti-NKG2DL-antibodies, apply the same strategy to the samples but note that ligand positivity lies in Alexa Fluor 488 and not the PE channel.</li>
	<li>Adjust gates according to the FMO controls for gating strategy from step 6.4 and 6.5 (see Table 3) for appropriate FMO controls to perform for this experiment: In the analysis software, draw a gate on the negative fraction. While recording the full-stained sample, the cells above the previously created gate are positive for the analyzed marker.</li>
</ol>

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<li>Eagle, R. A., Trowsdale, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promiscuity+and+the+single+receptor%3A+NKG2D%5BTitle%5D)+AND+%22Nature+Reviews.+Immunology%22%5BJournal%5D)">Promiscuity and the single receptor: NKG2D.</a> Nature Reviews. Immunology. 7 (9), 737-744 (2007).</li>
<li>El-Gazzar, A., Groh, V., Spies, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunobiology+and+conflicting+roles+of+the+human+NKG2D+lymphocyte+receptor+and+its+ligands+in+cancer%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Immunobiology and conflicting roles of the human NKG2D lymphocyte receptor and its ligands in cancer.</a> Journal of Immunology. 191 (4), 1509-1515 (2013).</li>
<li>Venkataraman, G. M., Suciu, D., Groh, V., Boss, J. M., Spies, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promoter+region+architecture+and+transcriptional+regulation+of+the+genes+for+the+MHC+class+I-related+chain+A+and+B+ligands+of+NKG2D%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Promoter region architecture and transcriptional regulation of the genes for the MHC class I-related chain A and B ligands of NKG2D.</a> Journal of Immunology. 178 (2), 961-969 (2007).</li>
<li>Long, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Negative+signaling+by+inhibitory+receptors%3A+the+NK+cell+paradigm%5BTitle%5D)+AND+%22Immunological+Reviews%22%5BJournal%5D)">Negative signaling by inhibitory receptors: the NK cell paradigm.</a> Immunological Reviews. 224, 70-84 (2008).</li>
<li>Hüe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+direct+role+for+NKG2D%2FMICA+interaction+in+villous+atrophy+during+celiac+disease%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease.</a> Immunity. 21 (3), 367-377 (2004).</li>
<li>Gasser, S., Raulet, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+and+self-tolerance+of+natural+killer+cells%5BTitle%5D)+AND+%22Immunological+Reviews%22%5BJournal%5D)">Activation and self-tolerance of natural killer cells.</a> Immunological Reviews. 214, 130-142 (2006).</li>
<li>Groh, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+stress-regulated+human+major+histocompatibility+complex+class+I+gene+expressed+in+gastrointestinal+epithelium%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (22), 12445-12450 (1996).</li>
<li>Maurer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet-mediated+shedding+of+NKG2D+ligands+impairs+NK+cell+immune-surveillance+of+tumor+cells%5BTitle%5D)+AND+%22Oncoimmunology%22%5BJournal%5D)">Platelet-mediated shedding of NKG2D ligands impairs NK cell immune-surveillance of tumor cells.</a> Oncoimmunology. 7 (2), 1364827(2018).</li>
<li>Paczulla, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absence+of+NKG2D+ligands+defines+leukaemia+stem+cells+and+mediates+their+immune+evasion%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.</a> Nature. 572 (7768), 254-259 (2019).</li>
<li>Paczulla, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+observation+reveals+high-frequency+engraftment+of+human+acute+myeloid+leukemia+in+immunodeficient+mice%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Long-term observation reveals high-frequency engraftment of human acute myeloid leukemia in immunodeficient mice.</a> Haematologica. 102 (5), 854-864 (2017).</li>
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The authors have nothing to disclose.

Here we present two flow cytometric methods that can detect NKG2DL surface expression on human primary AML cells. We show that both detection methods can be used in conjunction with other antibody stainings (e.g., detecting CD34 expression). Similar stainings may also be performed on other primary cell types and cell lines.
We recently showed that the absence of NKG2DL on the surface of AML patient blasts can enrich LSC14. In AML, NKG2DL negative but not NKG2DL positive...

NK cells are important effectors of the innate immune system that can recognize and eliminate malignant cells or stressed healthy cells (e.g., by a viral infection) without prior antigen stimulation1. This process is tightly regulated via a complex repertoire of activating receptors &#8212;such as natural cytotoxicity receptors (NCRs), NKG2D and CD16&#8212; and inhibitory receptors that are largely represented by killer immunoglobulin-like receptors (KIRs)2. Binding of KIRs to human leukocyte antigen (HLA) class I molecules on somatic cells ensures self-recognition and conveys NK cell tolerance. On the other hand, absence of self-recognition and increased binding of activating receptors to their ligands on the target cells trigger the release of cytotoxic granules leading to NK cell-mediated cytotoxicity1. Finally, NK cells can exert antibody-dependent cellular cytotoxicity (ADCC) by binding of the activating receptor CD16 to targets expressing the Fc portion of Ig (FcR)2. Apart from direct cytotoxicity, NK cells can also trigger cytokine release bridging the innate with the adaptive immune system3.
NKG2D is a major activating receptor expressed on NK, NKT, &#947;&#948; T, and na&#239;ve CD8+ T cells4 that enables such cytotoxic immune cells to recognize and lyse NKG2D ligand (NKG2DL) expressing target cells. Healthy cells commonly do not express NKG2DL. Instead, NKG2DL expression is upregulated on malignant or virus-infected cells to make these amenable to immune clearance5.
The human NKG2DL family comprises eight known molecules among which the two MHC I chain-related molecules A and B (MICA and MICB6) and the cytomegalovirus UL16-binding proteins 1&#8211;6 (ULBP1-67). The expression of NKG2DL is regulated on the transcriptional, post-transcriptional as well as the post-translational levels8. As such, while NKG2DL expression is commonly not detectable on the surface of healthy cells, NKG2DL mRNA9 and intracellular protein expression were reported in healthy tissues. The functional relevance of such expression and the mechanisms underlying such discrepant expression patterns remain to be defined10.
The mechanistic regulation of NKG2DL expression in the cancer cells is a fascinating area of investigation. Pathways known to be involved in either cellular stress, e.g., the heat shock stress pathway9, or DNA damage-associated pathways, such as the ataxia telangiectasia mutated (ATM) and Rad3 related (ATR) pathway11, as well as viral or bacterial infections have been directly linked to the induction of NKG2DL expression12. However, even if surface expression of NKG2DL has been effectively induced, this expression can be lost again through proteolytic-mediated shedding, a mechanism associated with immune escape and poor clinical prognosis in some cancers13.
The absence of cell surface NKG2DL may also play important roles in patients with AML. Here, treatment with intensive chemotherapy often induces remission, but relapse often occurs from leukemic stem cells (LSC), which selectively survive chemotherapies and evade immune response. As we recently showed, LSCs, for example, escape NK cell lysis by suppressing NKG2DL surface expression14.
Inversely, the absence of surface NKG2DL expression can be used as a method to identify and viably isolate putative stem-like subpopulations of cells from bulk counterpart leukemic subpopulations. Here, we present two flow cytometric approaches that can be used to detect NKG2DL surface expression and thereby identify NKG2DL negative stem cells in leukemia and perhaps also in other cancers: A method for pan-ligand surface recognition and a method involving staining with single or pooled antibodies recognizing individual known NKG2DL proteins.

<table><tbody><tr><td>7-amminoactinomycin D (7-AAD)</td><td>Invitrogen</td><td>A1310</td><td>Viability dye</td></tr><tr><td>96 well plate U bottom</td><td>Sarstedt</td><td>833925500</td><td>96 Well plate for our Flow cytometer</td></tr><tr><td>APC Mouse Anti-Human cd34</td><td>BD</td><td>555824</td><td>Antibody detecting CD34&lt;br/&gt; RRID: AB_398614</td></tr><tr><td>Bovine Serum Albumin</td><td>PanReac AppliChem</td><td>A1391,0050</td><td>Component of the staining buffer</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Roth</td><td>8043.1</td><td>Component of the staining buffer</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>BioConcept</td><td>2-01F10-I</td><td>Component of the supplemented RPMI medium</td></tr><tr><td>FlowJo 10.2</td><td>BD</td><td>/</td><td>Software enabling data analysis for flow cytometry experiment</td></tr><tr><td>Goat- anti-Rabbit IgG (H+L) Alexa Fluor 488</td><td>Thermo Scientific</td><td>A21222</td><td>Secondary antibody detecting the primary antibodies for MICA and MICB&lt;br/&gt; RRID: AB_1037853</td></tr><tr><td>Human NKG2D Fc Chimera Protein, CF</td><td>R&amp;amp;D</td><td>1299-NK-050</td><td>Fusion Protein detecting all NKG2DLs&lt;br/&gt; RRID:</td></tr><tr><td>Human ULBP-1 Antibody</td><td>R&amp;amp;D</td><td>AF1380</td><td>Antibody detecting ULBP1&lt;br/&gt; RRID: AB_354765</td></tr><tr><td>Human ULBP-2/5/6 Antibody</td><td>R&amp;amp;D</td><td>AF1298</td><td>Antibody detecting ULBP2/5/6&lt;br/&gt; RRID: AB_354725</td></tr><tr><td>Human ULBP-3 Antibody</td><td>R&amp;amp;D</td><td>AF1517</td><td>Antibody detecting ULBP3&lt;br/&gt; RRID: AB_354835</td></tr><tr><td>MICA Polyclonal Antibody</td><td>Thermo Scientific</td><td>PA5-35346</td><td>Antibody detecting MICA&lt;br/&gt; RRID: AB_2552656</td></tr><tr><td>MICB Polyclonal Antibody</td><td>Thermo Scientific</td><td>PA5-66698</td><td>Antibody detecting MICB&lt;br/&gt; RRID: AB_2663413</td></tr><tr><td>One-step Antibody Biotinylation Kit 1 strip, for 8 reactions</td><td>Miltenyibiotec</td><td>130-093-385</td><td>Biotinylation kit for the NKG2DL fusion protein</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma Aldrich</td><td>D8537-500ML</td><td>Component of the staining buffer</td></tr><tr><td>Rabbit anti-Goat IgG (H+L) Alexa Fluor 488</td><td>Thermo Scientific</td><td>A11034</td><td>Secondary antibody detecting the primary antibodies for the ULBPs&lt;br/&gt; RRID: AB_2576217</td></tr><tr><td>Rainbow Calibration Particles (8-peaks) 3.0 um</td><td>Spherotech Inc.</td><td>RCP-30-20A</td><td>Beads used for flow cytometry device maintainance</td></tr><tr><td>RPMI medium</td><td>Sigma Aldrich</td><td>R8758-500ML</td><td>Cell culture medium</td></tr><tr><td>RayBright Universal Compensation Beads</td><td>Raybiotech</td><td>137-00013-100</td><td>Beads used to create the compensation matrix</td></tr><tr><td>Streptavidin, R-Phycoerythrin Conjugate (SAPE) - 1 mg/mL</td><td>Invitrogen</td><td>S866</td><td>Seondary step for the biotinylated NKG2DL fusion protein detection</td></tr></tbody></table>

two flow cytometric approaches of nkg2d ligand surface detection to distinguish stem cells from bulk subpopulations in acute myeloid leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

Both the protocols presented here allow the enrichment of AML LSC by flow cytometric analyses using CD34, a known marker of LSC17, in combination with NKG2DL surface expression by either utilizing pan-ligand recognition or staining with pooled antibodies against individual ligands. In Figure 1, we show that the analyzed AML samples are positive for CD34 and NKG2DL, but negative subpopulations also exist, which is shown by the presence of four different populations in ...

Watch this Scientific Journal Video about Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia at JoVE.com

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

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Research

JoVE Journal

Cancer Research

4.1K Views.  University Hospital Basel and University of Basel. Dit protocol toont twee verschillende manieren om NKG2DLs op het oppervlak van AML-cellen te detecteren. De techniek biedt een snelle, gebruiksvriendelijke kleurmethode om alle bekende en mogelijk onbekende NKG2DLs te detecteren. Deze methode maakt het mogelijk om leukemische stamcellen te scheiden van bulk AML-cellen, waardoor het mogelijk is om deze cellen verder te karakteriseren.Begin met het ontdooien van de biotine en de NKG2D fusie-eiwitbuizen op kamertemperatuur. Draai snel het...

Video: Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Developmental Biology

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Neuroscience

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Immunology and Infection

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Medicine

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

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