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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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 
			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 
			RRID: AB_1037853</td>
		</tr>
		<tr>
			<td>Human NKG2D Fc Chimera Protein, CF</td>
			<td>R&#38;D</td>
			<td>1299-NK-050</td>
			<td>Fusion Protein detecting all NKG2DLs 
			RRID:</td>
		</tr>
		<tr>
			<td>Human ULBP-1 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1380</td>
			<td>Antibody detecting ULBP1 
			RRID: AB_354765</td>
		</tr>
		<tr>
			<td>Human ULBP-2/5/6 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1298</td>
			<td>Antibody detecting ULBP2/5/6 
			RRID: AB_354725</td>
		</tr>
		<tr>
			<td>Human ULBP-3 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1517</td>
			<td>Antibody detecting ULBP3 
			RRID: AB_354835</td>
		</tr>
		<tr>
			<td>MICA Polyclonal Antibody</td>
			<td>Thermo Scientific</td>
			<td>PA5-35346</td>
			<td>Antibody detecting MICA 
			RRID: AB_2552656</td>
		</tr>
		<tr>
			<td>MICB Polyclonal Antibody</td>
			<td>Thermo Scientific</td>
			<td>PA5-66698</td>
			<td>Antibody detecting MICB 
			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 
			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

4.0K Views.  University Hospital Basel and University of Basel. 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.

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 ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

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 ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

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 ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice.

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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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 ...