Name: standardization of transfer across labs between flow cytometers for detection of lymphocytes in japanese encephalitis vaccinated children
Uploaded: 2023-02-10T13:30:00.000+00:00
Duration: 6 min 22 s
Description: Watch this Scientific Journal Video about Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children at JoVE.com

RW was supported by Beijing Natural Science Foundation, China (No. 7222059), National Natural Science Foundation of China (No. 82002130), XZ was supported by the CAMS Innovation Fund for Medical Sciences (No. 2019-I2M-5-026).

The study was approved by the Ethics Committee of Beijing Children&#39;s Hospital, Capital Medical University (Approval Number: 2020-k-85). Informed consent of human subjects was waived as only residual samples after clinical testing were used in this study. Two labs are involved in this study. The transferring lab is where the standardized method was developed using one flow cytometer. The cytometer in this lab is hereinafter referred to as cytometer A. The test method lab is the laboratory that receives methods using another flow cytometer, and the cytometer in this lab is hereinafter referred to as cytometer B.
1. Peripheral blood sample collection and cell preparation
<ol>
	<li>Collect peripheral blood samples (2 mL) from JE-vaccinated children and unvaccinated children in EDTA-K2-anticoagulated tubes by standard venipuncture.</li>
	<li>Mix the whole blood sample by turning the tubes upside down 10x. Add 100 &#181;L of whole blood to a 12 mm x 75 mm tube. Make each sample in duplicate.</li>
	<li>Add 10 &#181;L of brilliant stain buffer (BV buffer) to a 12 mm x 75 mm tube. Add 5 &#181;L of each antibody (CD45, CD3, CD4, CD8, CD127, CD27, CD19, IgD; dilution factor: 1:20) and 20 &#181;L of antibody (CD25, CD45RA; dilution factor: 1:5) to the BV buffer. Vortex gently. 
	NOTE: The volumes and information of the antibody reagents are shown in Table 1.</li>
	<li>Add the antibody cocktail into the tube with the whole blood sample. Vortex gently and incubate at room temperature for 15 min in the dark.</li>
	<li>Dilute the 10x lysing solution 1:10 with distilled water to prepare 1x lysing solution. Lyse the blood samples by adding 2 mL of 1x lysing solution. Vortex gently and incubate for 10 min in the dark at room temperature.</li>
	<li>Centrifuge at 300 &#215; g for 5 min and decant the supernatant.</li>
	<li>Resuspend the pellet in 2 mL of 1x PBS, centrifuge at 300 &#215; g for 5 min, and decant the supernatant.</li>
	<li>Resuspend the pellet in 0.5 mL of 1x PBS and mix thoroughly. Store at 2 &#176;C to 8 &#176;C. Send the samples to the two labs for flow cytometric analysis. 
	&#8203;NOTE: The negative control sample and cell single-stained control must be processed simultaneously.</li>
</ol>
2. Preparing compensation beads and single-stained control
<ol>
	<li>Label V500-anti-CD45 single-stained, APC-H7-anti-CD3 single-stained, FITC-anti-CD4 single-stained, R718-anti-CD8 single-stained, BV605-anti-CD27 single-stained, APC-anti-CD45RA single-stained, PE-anti-CD25 single-stained, BV421-anti-CD127 single-stained, BB700-anti-CD19 single-stained, and PE-CY7-anti-IgD single-stained tubes.</li>
	<li>Add 100 &#181;L of 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) buffer containing 1% fetal bovine serum (FBS) to a 5 mL round-bottom polystyrene test tube.</li>
	<li>Add 50 &#181;L of the negative beads and 50 &#181;L of the anti-mouse Ig, &#954; beads to each tube and vortex.</li>
	<li>Add 2 &#181;L of each labeled antibody (CD45, CD3, CD4, CD8, CD25, CD127, CD45RA, CD27, CD19, IgD) to a single-stained tube. Vortex the mixture gently and incubate for 15 min in the dark at room temperature (20 &#176;C to 25 &#176;C).</li>
	<li>Resuspend the beads in 2 mL of 1x PBS, centrifuge at 300 &#215; g for 5 min, and decant the supernatant.</li>
	<li>Resuspend the beads in 0.5 mL of 1x PBS and mix thoroughly. Store at 2 &#176;C to 8 &#176;C.</li>
</ol>
3. Using identical configuration names on the different cytometers in two labs
<ol>
	<li>Create a new configuration in the software of cytometer A. Click the Cytometer button and choose view Configurations to display the configuration window.</li>
	<li>In the configuration list, right-click the base configurations folder, choose New Folder, and rename it.</li>
	<li>Right-click the base configuration and choose copy.</li>
	<li>Right-click the new folder and paste the base configuration to create a new configuration. Rename the new configuration &#34;Standardized Method Transfer&#34;.</li>
	<li>Drag the parameter name, such as FITC, from the Parameters list onto the appropriate detector (530/30).</li>
	<li>Edit additional parameters (PE, BB700, PE-CY7, APC, R718, APC-H7, BV421, V500, and BV605) to the new configuration.</li>
	<li>Follow this method to create a new configuration on a different flow cytometer model using the same name &#34;Standardized Method Transfer&#34;. Ensure that the configuration includes the same parameters for each detector. 
	NOTE: To successfully identify the experiment template on the two different flow cytometer models, make sure that the name is consistent.</li>
	<li>Under the cytometer configurations, use cytometer setup and tracking (CST) beads to define the cytometer baseline and track cytometer performance. Click on the Cytometer button and choose CST to display the cytometer setup and tracking workspace.</li>
	<li>Add three drops (150 &#181;L) of setup beads to 0.5 mL of 1x PBS in a 5 mL round-bottom polystyrene test tube. Place the beads tube on the cytometer.</li>
	<li>In the Setup Control window, choose Define Baseline, and click Run.</li>
</ol>
4. Standardizing experiment using cytometer A in the transferring lab
<ol>
	<li>Run a performance check using the cytometer setup and tracking beads to verify that the cytometer is performing well.</li>
	<li>In the software browser window, right-click the cytometer settings and choose Application Settings to create a worksheet.</li>
	<li>Using an unstained sample, adjust the photomultiplier tube (PMT) voltages of FSC, SSC, and all fluorescence parameters. FSC: 260; SSC: 460; FITC: 490; PE: 575; BB700: 620; PE-CY7: 640; APC: 600; R718: 620; APC-H7: 620; BV421: 466; V500: 420; and BV605: 505.</li>
	<li>Right-click the experiment cytometer settings and choose application settings to save it.</li>
	<li>Click on the experiment button and choose compensation setup menu. Then, choose Create compensation controls to automatically add compensation controls.</li>
	<li>Record data for all compensation control beads.</li>
	<li>Click on the experiment and choose compensation setup. Then, calculate the compensation automatically.</li>
	<li>Record data for cell single-stained controls.</li>
	<li>Use CD45RA APC to modify the compensation value of R718 to 15% APC. Use CD19 BB700 to modify the compensation value of APC-H7 to 14% BB700. Use CD8 R718 to modify the compensation value of APC-H7 to 60% R718.</li>
	<li>Record data for the CST beads.</li>
	<li>Create a global worksheet of the target value template for the CST bright beads.</li>
	<li>Using an FSC/SSC plot, draw the polygon gate for the CST bright beads. Obtain histogram plots of 10 fluorescence channels for the CST bright beads: FITC, PE, BB700, PE-Cy7, APC, R718, APC-H7, BV421, V500, and BV605. Create interval gates in the histogram plots. Create the statistics view by showing the median of the interval gates.</li>
	<li>Run the samples on the flow cytometer; collect a total of 25,000 lymphocytes.</li>
	<li>Create a global worksheet of the analysis template for the samples.</li>
	<li>Use the following sample gating strategy:
	<ol>
		<li>Using a CD45/side scatter-area (SSC-A) dot plot, draw the polygon gate to identify the intact lymphocyte population while excluding debris.</li>
		<li>Using a CD3/CD19 dot plot, draw a rectangular gate to select CD3+ T cells and CD19+ B cells.</li>
		<li>Using a CD4/CD8 dot plot, draw a quad gate to select CD4+ or CD8+ T cells (cells with a high fluorescence for these markers, respectively).</li>
		<li>Using a CD45RA/CD27 dot plot, draw a quad gate to subdivide CD8+ or CD4+ T cells into na&#239;ve (CD27+CD45RA+), TCM (CD27+ CD45RA-), TEM (CD27-CD45RA-), and TEMRA (CD27-CD45RA+) cells.</li>
		<li>Using a CD25/CD127 dot plot, draw a quad gate to subdivide CD4+ T cells into Treg cells (CD4+CD25++CD127low).</li>
		<li>Using a CD27/IgD dot plot, draw a quad gate to subdivide CD19+ B cells into na&#239;ve B cells (CD27-IgD+) and memory B cells (CD27+IgD-).</li>
	</ol>
	</li>
	<li>Save the experimental template on cytometer A.</li>
	<li>Use the CD-ROM to export the experimental template.</li>
</ol>
5. Transferring the experimental template to cytometer B in the test method lab
NOTE: The experimental template includes instrument settings, the analysis template, and the target value template of median fluorescence intensity (MFI).
<ol>
	<li>Conduct a performance check using CST beads to verify that the cytometer is performing well.</li>
	<li>Import the experimental template and create an experiment for cytometer B using the template.</li>
	<li>Use the same lot of CST beads to adjust the fluorescent parameter voltages for each fluorescence channel to match the previous MFI instrument. 
	NOTE: The MFI values vary within &#177;5% (the MFI of bright beads before and after transfer are shown in Table 2).</li>
	<li>Adjust the voltage for FSC using the unstained sample, if needed.</li>
	<li>Right-click on the experimental cytometer settings and choose application settings to save the application settings.</li>
	<li>Use CD45RA APC to modify the compensation value of R718 to 15% APC. Use CD19 BB700 to modify the compensation value of APC-H7 to 24% BB700. Use CD8 R718 to modify the compensation value of APC-H7 to 60% R718.</li>
	<li>Run the samples on the flow cytometer; collect a total of 25,000 lymphocytes.</li>
</ol>
6. Consistency between the experimental results obtained on the two cytometers
<ol>
	<li>Assess the differences in lymphocyte subsets between instruments using one-way ANOVA (p &#60; 0.05).</li>
</ol>

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	<li>Cabanski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+method+transfer:+Recommendations+for+best+practice.">Flow cytometric method transfer: Recommendations for best practice.</a> Cytometry Part B. Clinical Cytometry. 100 (1), 52-62 (2021).</li><li>Le Lann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+procedure+for+flow+cytometry+data+harmonization+in+prospective+multicenter+studies.">Standardization procedure for flow cytometry data harmonization in prospective multicenter studies.</a> Scientific Reports. 10 (1), 11567 (2020).</li><li>Linskens, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+standardization+of+flow+cytometry+diagnostic+screening+of+primary+immunodeficiency+by+software-based+automated+gating.">Improved standardization of flow cytometry diagnostic screening of primary immunodeficiency by software-based automated gating.</a> Frontiers in Immunology. 11, 584646 (2020).</li><li>Glier, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+8-color+flow+cytometry+across+different+flow+cytometer+instruments:+A+feasibility+study+in+clinical+laboratories+in+Switzerland.">Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.</a> Journal of Immunological Methods. 475, 112348 (2019).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+and+disease+burden+of+children+hospitalized+for+viral+infections+within+the+family+Flaviviridae+in+China:+A+national+cross-sectional+study.">The epidemiology and disease burden of children hospitalized for viral infections within the family Flaviviridae in China: A national cross-sectional study.</a> PLOS Neglected Tropical Diseases. 16 (7), e0010562 (2022).</li><li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+values+for+peripheral+blood+lymphocyte+subsets+of+healthy+children+in+China.">Reference values for peripheral blood lymphocyte subsets of healthy children in China.</a> The Journal of Allergy and Clinical Immunology. 142 (3), 970-973 (2018).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+polyfunctional+T+cells+in+children+vaccinated+with+Japanese+encephalitis+vaccine+via+the+flow+cytometry+technique.">Detection of polyfunctional T cells in children vaccinated with Japanese encephalitis vaccine via the flow cytometry technique.</a> Journal of Visualized Experiments. (187), e64671 (2022).</li><li>Yu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)+CD25+(+)+CD127+(low/-)+T+cells:+a+more+specific+Treg+population+in+human+peripheral+blood.">CD4(+) CD25 (+) CD127 (low/-) T cells: a more specific Treg population in human peripheral blood.</a> Inflammation. 35 (6), 1773-1180 (2012).</li><li>Kalina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+flow+cytometry+through+standardization:+opportunities+and+challenges.">Reproducibility of flow cytometry through standardization: opportunities and challenges.</a> Cytometry Part A. 97 (2), 137-147 (2020).</li><li>Sommer, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+highly+sophisticated+flow+cytometry+assays+in+multicenter+clinical+studies:+considerations+and+guidance.">Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance.</a> Bioanalysis. 7 (10), 1299-1311 (2015).</li><li>Glier, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comments+on+EuroFlow+standard+operating+procedures+for+instrument+setup+and+compensation+for+BD+FACS+Canto+II,+Navios+and+BD+FACS+Lyric+instruments.">Comments on EuroFlow standard operating procedures for instrument setup and compensation for BD FACS Canto II, Navios and BD FACS Lyric instruments.</a> Journal of Immunological Methods. 475, 112680 (2019).</li></ol>

The authors declare no conflicts of interest.

Immunophenotyping of peripheral blood lymphocyte subsets can help understand the changes in cell-mediated adaptive immunity after vaccination in children. In clinical applications, unexpected situations occur, such as a failure to detect samples in a timely manner or the replacement of a flow cytometer; therefore, rapid standardized methods that facilitate transfers between flow cytometers in different labs are needed9,10,11. He...

The standardization of flow cytometry is useful for the comparability of results obtained from different cytometers across different laboratories and study centers, and conducive to the mutual recognition of results to improve work efficiency. An increasing number of scenarios require standardization. During the drug development process, flow cytometry standardization is important, as a developed and validated assay will support the whole drug development process from preclinical to clinical analysis. Flow cytometric methods are frequently transferred between the pharmaceutical industry and collaborating laboratories1. Moreover, it is essential to obtain comparable data from multicentric clinical studies. For example, a standardization workflow was developed in the Systemic Autoimmune Diseases Multicenter Clinical Research Project to obtain comparable data from multicentric flow cytometry2.
The standardization of flow cytometry methods is challenging. The challenges experienced across labs are attributed to the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations among different flow cytometers and divergent gating strategies between the centers3,4. Therefore, it is important to conduct a gap analysis between laboratories. Sample access, quality systems, personnel qualifications, and instrument configuration must be reviewed to ensure that the requirements are met.
At present, children vaccinated with the Japanese encephalitis (JE) vaccine have a significantly reduced incidence of JE5. Monitoring peripheral blood immune cells can help understand the changes in cell-mediated adaptive immunity after vaccination, and the correlation between the changes in peripheral blood lymphocyte subsets and the effects of vaccination. Due to the limited stability of whole blood samples, evaluations of vaccine efficacy are often performed in multiple centers. For this analysis, we defined na&#239;ve CD8+ or CD4+ T cells as CD27+ CD45RA+, central memory T cells (TCM) as CD27+ CD45RA-, effector memory T cells (TEM) as CD27- CD45RA-,&#160;and terminally differentiated effector memory T cells (TEMRA) as CD27- CD45RA+. CD19+ B cells can be separated into populations that express CD27 versus IgD6,7, na&#239;ve B cells express CD27n memory B cells (mBCs) can be identified based on the expression of IgD6, and regulatory T cells (Tregs) can be identified as CD4+CD25++CD127low 8. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results in multiple centers, a rapid and feasible standardization method was established to facilitate the transfer of protocols across different flow cytometers for the detection of lymphocytes in the whole blood of JE-vaccinated children. Six healthy children (2 years old) were recruited from Beijing Children&#39;s Hospital, Capital Medical University. After receiving a prime and boost vaccination with a live-attenuated JE SA14-14-2 vaccine less than 6 months prior, peripheral blood samples were collected from the volunteers. Highly comparable data were obtained from different instruments following standardized procedures, which is helpful for multicenter assessments.

<table><tbody><tr><td>BD CompBeads Anti-Mouse Ig, &amp;kappa;/Negative Control Compensation Particles Set</td><td>BD</td><td>552843</td><td>compensation</td></tr><tr><td>BD FACSCanto</td><td>BD</td><td>FACSCanto</td><td>flow Cytometry A in the transferring lab</td></tr><tr><td>BD FACSDiva CS&amp;amp;T Research Beads</td><td>BD</td><td>655051</td><td>define flow cytometer baseline and track cytometer performance</td></tr><tr><td>BD Horizon BV421 Mouse Anti-Human CD127</td><td>BD</td><td>562436</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD Horizon BV605 Mouse Anti-Human CD27</td><td>BD</td><td>562656</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD Horizon V500 Mouse Anti-Human CD45</td><td>BD</td><td>560777</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD LSRFortessa</td><td>BD</td><td>LSRFortessa</td><td>flow Cytometry B in the test method lab</td></tr><tr><td>BD OptiBuild BB700 Mouse Anti-Human CD19</td><td>BD</td><td>745907</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD OptiBuild R718 Mouse Anti-Human CD8</td><td>BD</td><td>751953</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD Pharmingen APC Mouse Anti-Human CD45RA</td><td>BD</td><td>550855</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD Pharmingen APC-H7 Mouse Anti-Human CD3</td><td>BD</td><td>560176</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD Pharmingen FITC Mouse Anti-Human CD4</td><td>BD</td><td>566320</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD Pharmingen PE Mouse Anti-Human CD25</td><td>BD</td><td>555432</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>BD Pharmingen PE-Cy7 Mouse Anti-Human IgD</td><td>BD</td><td>561314</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>Brilliant Staining Buffer Plus</td><td>BD</td><td>566385</td><td>Staining Buffer</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810</td><td>Cell centrifugation</td></tr><tr><td>Centrifuge Tube</td><td>BD Falcon</td><td>BD-35209715</td><td>15 mL centrifuge tube</td></tr><tr><td>CS&amp;amp;T IVD Beads</td><td>BD</td><td>662414</td><td>standard beads to setup cytometer settings in different flow cytometer</td></tr><tr><td>Lysing Solution 10x Concentrate</td><td>BD</td><td>349202</td><td>lysing red blood cells</td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>Gibco</td><td>10010-023</td><td>PBS</td></tr><tr><td>Round-bottom test tube</td><td>BD Falcon</td><td>352235</td><td>5 mL test tube</td></tr></tbody></table>

standardization of transfer across labs between flow cytometers for detection of lymphocytes in japanese encephalitis vaccinated children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

Figure 1&#160;shows a global worksheet of the target value template for the CST bright beads. Using an FSC/SSC plot, a polygon gate is drawn to select the CST bright beads. Histogram plots of 10 fluorescence channels were obtained for the CST bright beads: FITC, PE, BB700, PE-Cy7, APC, R718, APC-H7, BV421, V500, and BV605. The target value for each parameter is displayed by showing the median within the histogram gates in Table 2. The screenshots of templates and parameter s...

Watch this Scientific Journal Video about Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children at JoVE.com

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...

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JoVE Journal

Immunology and Infection

914 Views.  Becton Dickinson Medical Devices (Shanghai) Co Ltd. De standaardisatiemethode van de flowcytometer biedt een haalbare aanpak voor de wederzijdse accreditatie van laboratoriumresultaten en zorgt voor de consistentie van resultaten met onderzoeksprojecten in meerdere centra. De methode minimaliseert het tijdsverbruik is eenvoudig uit te voeren, is uitgerust om te automatiseren, analyseert sjabloon en kan de vereiste van gegevensanalyse drastisch verminderen. Begin met het maken van een nieuwe configuratie in de software van cytometer A, vanuit h...

Video: Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

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This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

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Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

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This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has ...

Biochemistry

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Developmental Biology

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

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. ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" of a T cell needs to be assessed by function, and higher function is associated with more powerful immune protection. T cells that can simultaneously produce two or more cytokines or chemokines at the single-cell level are called polyfunctional T cells (TPFs), which mediate immune responses through a variety of molecular mechanisms to express degranulation markers (CD107a) and secrete interferon (IFN)-&#947;, tumor necrosis factor (TNF)-&#945;, interleukin (IL)-2, or macrophage inflammatory protein (MIP)-1&#945;. There is increasing evidence that TPFs are closely related to the maintenance of long-term immune memory and protection and that their increased proportion is an important marker of protective immunity and is important in the effective control of viral infection and reactivation. This evaluation applies not only to specific immune responses but also to the assessment of cross-reactive immune responses. Here, taking the Japanese encephalitis virus (JEV) as an example, the detection method and flow cytometry color scheme of JEV-specific TPFs produced by peripheral blood mononuclear cells of children vaccinated against Japanese encephalitis were tested to provide a reference for similar studies.

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (TPF)&#160;profiles in peripheral blood mononuclear cells (PBMCs) within Japanese encephalitis virus (JEV)-vaccinated children. The detection method and flow cytometry color scheme of JEV-specific TPFs were tested to provide a reference for similar studies.

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

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