Name: quality-controlled sputum analysis by flow cytometry
Uploaded: 2021-08-09T13:00:00.000+00:00
Duration: 7 min 22 s
Description: Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

We want to thank David Rodriguez for his assistance with the figure preparation. Sputum samples were run on the BD LSR II at the UT Health San Antonio Flow Cytometry Shared Resource Facility, supported by UT Health, NIH-NCI P30 CA054174-20 (CTRC at UT Health) and UL1 TR001120 (CTSA grant).

All steps of the sputum processing are performed in a biological safety cabinet with appropriate personal protective equipment.
1. Reagent preparation before starting sputum dissociation
<ol>
	<li>Thaw 1% Paraformaldehyde (PFA), 25 mL per sample on ice, and keep cold until use. 
	CAUTION: PFA is toxic by inhalation and skin contact. Prepare the fixative according to the manufacturer&#39;s instructions and freeze at -20 &#176;C in 25 mL aliquots until use.</li>
	<li>Approximate the weight of the sample and thaw enough 0.1% Dithiothreitol (DTT) for step 2.2 and bring it to 37 &#176;C. (Aliquots of DTT should be stored at -20 &#176;C before use.)</li>
	<li>Bring enough 0.5% N-acetyl- L-cysteine (NAC) up to 37 &#176;C for step 2.2. (NAC should be made fresh weekly and stored at 4 &#176;C before use.)</li>
</ol>
2. Sputum dissociation
<ol>
	<li>Weigh the sputum sample to determine the volumes of dissociation reagents. 
	NOTE: A sample is considered to be small if the initial weight is &#8804;3 g, medium if &#62;3 g but &#8804;8 g, large if &#62;8 but &#8804;16 g, and extra-large if a sample weighs &#62;16 g. The indications small, medium, large, and extra-large will be used throughout the protocol. The quantity of reagents required for dissociation and labeling differ according to the size of the sputum sample.</li>
	<li>Transfer a small sample to a clean 50 mL conical tube, a medium sample to a clean 250 mL plastic disposable bottle, or a large and extra-large sample to a clean 500 mL plastic disposable bottle. Add 1 mL/g sample weight of 0.5% NAC and 4 mL/g sample weight of 0.1% DTT.</li>
	<li>Vortex at maximum speed (for 15 s), and then rock at room temperature (at maximum speed) for 15 min.</li>
	<li>Dilute the sample with four volumes of Hank&#39;s Balanced Salt Solution (HBSS) (based on the total volume of sample + reagents) to neutralize the NAC and DTT; vortex quickly at maximum speed and rock at room temperature for 5 min at maximum speed.</li>
	<li>Filter the cell suspension through 100 &#181;m nylon mesh cell strainer(s) into one or more 50 mL conical centrifuge tube(s) to create a single-cell suspension.</li>
	<li>Centrifuge the cells at 800 x g for 10 min at 4 &#176;C. Aspirate the supernatant, combine all pellets in one 15 mL conical tube, and then wash the pellets with HBSS using the same conditions.</li>
	<li>Resuspend the cell pellet in a volume of buffer determined by the initial weight of the sputum sample. 
	NOTE: A small sample is resuspended in 250 &#181;L of HBSS. A medium sample is resuspended in 760 &#181;L of HBSS. Large and extra-large samples are resuspended in 1460 &#181;L of HBSS.</li>
	<li>Take an aliquot of the cell suspension for a live/dead cell count using Trypan Blue. 
	NOTE: For a small sample, use 5 &#181;L. For a medium, large or extra-large sample, use 10 &#181;L. Dilute 1:10 with HBSS.
	<ol>
		<li>Mix 10 &#181;L of the sputum dilution with 30 &#181;L of 0.4% Trypan Blue to achieve a final sample dilution of 1:40. Load into the counting chambers of a hemocytometer for a cell count. 
		NOTE: It may be necessary to adjust the final dilution if the cell numbers are too low or too high to achieve an accurate count. Consulting Guiot et al.20 for correctly distinguishing sputum cells from SECs and debris is strongly recommended here. This is essential for an accurate cell count.</li>
	</ol>
	</li>
	<li>From the extra-large sample, remove 50 x 106 cells from the total and add to a new tube with enough added HBSS to create a total volume of 1700 &#181;L. 
	&#8203;NOTE: Consider this a large sample for the remainder of the protocol. Leftover samples can be discarded or used for other purposes.</li>
</ol>
3. Antibody and viability dye staining
<ol>
	<li>Choice of antibody and staining dye 
	NOTE: Table 1 shows the antibodies and viability dye that are used in this protocol and the cell populations they identify.
	<ol>
		<li>Label the tubes containing the sputum cells (see Table 2 for labels).</li>
		<li>Use 5 mL flow cytometry tubes (compatible with the flow cytometer used) for the sample tube with the unstained cells and the tube with the isotype control. Use 15 mL conical tubes for the blood and epithelial tube samples. 
		NOTE: These samples will be transferred to flow cytometry tubes following antibody staining and fixation.</li>
	</ol>
	</li>
	<li>Label the compensation tubes (Table 3). 
	NOTE: Use 5 mL flow cytometry tubes compatible with the flow cytometer being used.</li>
	<li>Add the amount of HBSS, antibody and/or dye to each of the sputum cell tubes and compensation tubes as indicated in Table 2 and Table 3, respectively. 
	NOTE: Add buffer (HBBS), antibodies, and dye to all the tubes before adding cells or compensation beads to ensure that all tubes&#39; staining time is consistent.</li>
	<li>Add the amounts of sputum cell volume listed in Table 2 to the assay tubes.</li>
	<li>Add compensation beads to the compensation tubes as listed in Table 3.</li>
	<li>Incubate all the tubes (assay and compensation tubes) on ice, protected from light, for 35 min. Then, fill the tubes with ice-cold HBSS and centrifuge at 4 &#176;C for 10 min at 800 x g.</li>
	<li>For the compensation tubes, aspirate the supernatant as close to the pellets as possible and flick the pellets to loosen.</li>
	<li>Add 0.5 mL of cold HBSS to the compensation tubes, store them on the ice at 4 &#176;C and protect them from light until needed for flow cytometry analysis.</li>
	<li>Aspirate the supernatant from the unstained isotype, blood, and epithelial tubes after centrifugation (step 3.6 from the antibody staining section) and loosen the pellets by flicking the tubes.</li>
</ol>
4. Fixation with 1% Paraformaldehyde (PFA)
<ol>
	<li>Add cold 1% PFA (which should be thawed by now) to the unstained, isotype, blood, and epithelial tubes; 2 mL to the unstained and isotype tubes, and 10 mL to the blood and epithelial tubes.</li>
	<li>Incubate the tubes on ice, protected from light for 1 h. Vortex quickly at maximum speed after 30 min.</li>
	<li>Fill the tubes with ice-cold HBSS. Then, centrifuge tubes at 4 &#176;C for 10 min at 1600 x g.</li>
	<li>Aspirate as much supernatant as possible without disturbing the cell pellet and flick the tube with the fingers to loosen the cells.</li>
	<li>Add 200 &#181;L of cold HBSS to the unstained and isotype tubes.</li>
	<li>Calculate the volume of HBSS for resuspension of the blood and epithelial tube according to the total cell count. 
	NOTE: Resuspension volume = 0.15 x [total cell count (step 8 of sputum dissociation) / 106]. Use 50 x 106 as the cell count for an extra-large sample.</li>
	<li>Store all sample and compensation tubes, protected from light on the ice at 4 &#176;C until flow cytometry analysis is performed. 
	&#8203;NOTE: This protocol has not been tested for storage for more than 24 h.</li>
</ol>
5. Data acquisition on the flow cytometer
<ol>
	<li>Apply appropriate startup procedures for the flow cytometer being used. 
	NOTE: This section of the protocol assumes that the person operating the flow cytometer is trained in the use of the instrument available to them, especially concerning daily procedures including checking the stability of the optics and fluidics systems, techniques for standardizing light scatter and fluorescence intensity, as well as calculating and applying the correct compensation matrix.</li>
	<li>Use the mixture of National Institute of Standards and Technology (NIST) beads to ensure that forward scatter and side scatter voltages are set to place the NIST beads to span the entire plot without placing the beads too close to the axes. 
	NOTE: This step is crucial to ensure that debris smaller than 5 &#181;m can be eliminated by gating post-acquisition analysis. Depending on the flow cytometer used, be mindful that the smallest beads are not being excluded with the threshold (when using the LSR II or the Lyric) or with the high discriminator (using the Navios EX flow cytometer). For the Navios EX, a gain of 2 was used for the forward and side scatter, a voltage of 236 for forward scatter, and 250 for side scatter. For the LSR II, a forward scatter voltage of 165 and a side scatter voltage of 190 was used.</li>
	<li>Set the flow rate to medium (LSR II) or high (Navios EX). 
	NOTE: The medium or high flow rate for most instruments can be used to acquire the sputum tubes. It is important to note that using too slow of a flow rate or too dilute of the sample may result in the cells settling, resulting in increased vortexing which is not desired. Therefore, adhering to the calculated resuspension volume in step 4.6 should result in cellular density that can be acquired quickly but not clog the machine.</li>
	<li>Adjust the voltages for each parameter used for the scatter and fluorescent parameters to place the cell populations on the scale. Use the figures with the gating strategy as guidance on how to adjust the voltages accordingly. 
	NOTE: Ensure that all the parameters needed are selected in the parameter selection window before the acquisition or that data will not be acquired.</li>
	<li>Acquire data for the unstained sputum sample first, followed by the isotype-stained sample, and then the blood tube and the epithelial tube. 
	NOTE: If the cell suspension is too concentrated to allow the flow cytometer to run the samples without clogging, the samples may be further diluted with HBSS.</li>
</ol>

All the authors are past or current employees of bioAffinity Technologies.

The cellular content of sputum includes a large variety of wide-ranging cells, often accompanied by a lot of debris37. In addition, sputum analysis requires a quality control that confirms the sample is collected from the lung instead of the oral cavity38. Therefore, it is not as simple to analyze sputum by flow cytometry as it is for blood, for example, which releases a much cleaner and homogeneous cell suspension. This protocol has addressed all these issues: providing instrument settings using specific size beads to ensure that both the smallest and largest cell populations can be detected, a gating strategy to eliminate debris, cell clumps, contaminating SECs and other dead cells, and lastly, a quality control measure for ensuring a sputum sample is from the lung instead of being mostly saliva.
There are critical steps in the protocol in working with sputum that is worth pointing out. First, cell yield can be drastically impacted by the number of nylon cell strainers used in step 2.5 of the sputum dissociation part of the protocol. Multiple strainers may be required to avoid losing too many cells due to clogging of the strainer. When the flow through the strainers has slowed down noticeably, a new strainer should be used. Second, the cell pellet of sputum samples can be very loose, especially if there is high contamination of SECs. Therefore, it is essential not to aspirate too close to the pellet when removing the supernatant after centrifuging the samples. Aspirating too close to the pellet may result in cell loss, if not loss of the entire pellet. Third, this protocol requires a fixation step. This preserves the cells and their staining profile and serves as a safety measure to protect the flow cytometer operator. Running samples on certain flow cytometers may present increased biological hazards due to the potential for aerosol production39. The PFA fixation helps to protect the operator from potential pathogens in the sputum sample. Fourth, and perhaps the most challenging aspect of preparing sputum samples for flow cytometric analysis, is cell counting. Cell counting is complicated because of the large variety of cell types present in the sputum. Counting machines are often limited by the cell size range they can capture and are therefore less reliable than a hemocytometer. However, cell counting of sputum samples by hemocytometer, which is excellently described by Guiot et al.20, is tedious and requires practice to become proficient. An accurate cell number is vital in determining the sample&#39;s final resuspension volume to allow for a reasonable flow rate and prevent clogs in the flow cytometer. The presence of the very large SECs in the sputum and the many smaller cell clumps that are not broken up by the dissociation buffer, nor caught by the filters, increase the likelihood of clogging the flow cytometer. Therefore, it is recommended to spend some time determining the best cell concentration/flow rate for data acquisition using the available flow cytometer. Additionally, ensure that the flow cytometer has the appropriate flow cell and nozzle size (or probe) capable of measuring the bigger cell populations present in the sputum.
Even when proficiency in cell counting has been achieved, it is still a time-consuming process. Therefore, the reagents&#39; determination for cell labeling in this protocol is based on the sample size (as judged by weight) rather than cell number. This allows for more efficient use of time since the manual cell count can be completed during the time needed for the antibody and viability dye staining. However, there are three notable exceptions to this. If a sample is &#62;16 g (the so-called extra-large samples), the manual cell count should occur before staining so that expensive reagents can be conserved by only staining 25 x 106 cells each for the blood and epithelial tubes. This cell number gives very reliable profiles in the settings described in this protocol. Another potential exception is the case of a very small sample. The estimation is that a minimum of 1-2 x 106 cells is needed for the flow cytometry analysis as presented in this protocol to generate reliable profiles (data not shown). Therefore, if a sample contains fewer than 1-2 x 106 cells, it may not be worth continuing with the procedure since the final result will likely be unacceptable.
Specific labeling reagents that work well on peripheral blood cells or cell lines may work very differently on blood cells contained in sputum (unpublished observations). Therefore, it is recommended to titrate each antibody or other labeling reagents according to well-established protocols34,35 before they are used in flow cytometry experiments. Most antibody titrations should give comparable results when staining up to 10 to 50-fold; however, it is advised to do additional titrations with cell numbers higher than that range34. When a reagent concentration has been determined, it is essential to keep the incubation time of that reagent the same as in the actual experiment. For that reason, the protocol stresses adding all the buffer and reagents to the tubes before&#160;the cells or compensation beads are added. This order of adding reagents and cells/beads together allows for higher consistency in labeling times. If, for some reason, the experimental incubation time cannot be kept similar to that used for antibody/dye titration, the latter should be repeated with an incubation time that is feasible during experiments.
Sputum samples have been widely used as a diagnostic specimen to study the pathophysiology of various illnesses affecting the lung. Tuberculosis40,41, COPD13,42, asthma43, cystic fibrosis44,45, lung cancer46,47,48, and rheumatoid arthritis49 are among the many illnesses where sputum has been studied due to the advantage of it being a non-invasively obtained specimen. More recently, during the coronavirus pandemic, sputum has been used to detect prolonged viral shedding in patients hospitalized with coronavirus disease 2019 (COVID-19)50.
Various technologies, including reverse transcription-polymerase chain reaction (RT-PCR), protein analysis, microscopy, and flow cytometry, have been used to identify disease-specific markers in sputum. Using a flow cytometric platform to analyze sputum samples is not widely used, but its use increases. With recent advancements in the technology of flow cytometers3,51,52, as well as advancements in the chemistries of fluorophores, generation of new antibody clones, and development of various dyes to identify dead cell populations51,53,54, the possibility of designing antibody panels aimed at diagnosing human diseases has dramatically increased. Moreover, the recent push for automating the analysis of flow cytometric data55,56 will eliminate the potential bias in manually reading and interpreting flow data. These new developments in flow cytometry will significantly facilitate the exploration of sputum as a clinical diagnostic.

Technical advancements in the hardware and software of flow cytometers have made it possible to identify many distinct cell populations simultaneously1,2,3,4. The utilization of the flow cytometer in hematopoietic cell research, for example, has led to a much better understanding of the immune system2 and the cellular hierarchy of the hematopoietic system5, as well as the diagnostic distinction of a multitude of different blood cancers6,7,8. Although most sputum cells are of hematopoietic origin9,10,11, flow cytometry has not been widely applied to sputum analysis for diagnostic purposes. However, several studies suggest that the evaluation of immune cell populations in sputum (the most significant subset of cells) may be of great help in diagnosing and/or monitoring diseases such as asthma and chronic obstructive pulmonary disease (COPD)12,13,14,15. Moreover, the existence of epithelial-specific markers that can be used in flow cytometry allows the interrogation of the following most significant subset of cells in sputum, lung epithelial cells.
In addition to the ability to analyze many distinct cell populations of different tissue origins, a flow cytometer can evaluate large numbers of cells in a relatively short period. In comparison, slide-based, cytological types of analyses often require highly specialized personnel and/or equipment. These analyses can be labor-intensive, which leads to only a proportion of the sputum sample being analyzed16.
Three critical issues limit the widespread use of sputum in flow cytometry. The first issue relates to the collection of sputum. Sputum is collected through a huff cough that expels mucus from the lungs into the oral cavity, subsequently spitting into a collection cup. Since the mucus travels through the oral cavity, there is a high chance of SEC contamination. This contamination complicates the specimen analysis, but the problem is easily rectified on a flow cytometric platform, as shown in this study.
Not everyone can produce sputum spontaneously; therefore, several devices have been developed to assist with the sputum collection in a non-invasive manner17. The nebulizer is one such device and has been shown to produce reliable sputum samples18,19,20. Although the nebulizer is a very effective way of non-invasively collecting sputum, its use still requires a setting at a medical facility with specialized personnel21. In contrast, handheld devices such as the lung flute22,23,24 and the acapella16,25 can be used at home since they are very user-friendly. These assist devices are both safe and cost-effective.
For us, the acapella gave consistently better results than the lung flute16, and therefore, the acapella device has been chosen for sputum collections. A 3-day collection sample was decided because the primary purpose for using sputum is to develop a lung cancer detection test16. It has been shown that a 3-day sample increases the likelihood of lung cancer detection compared to a 1- or 2-day sample26,27,28. However, other methods of sputum collection may be preferable for different purposes. If a different sputum collection method is used than the one described here, it is recommended to carefully titrate each antibody or dye used for flow cytometric analysis; very little data is available on how different sputum collection methods affect the targeted proteins for flow cytometry.
The second issue dampening the enthusiasm for using sputum for diagnostics, primarily related to flow cytometry, is cell number. The problem is the collection of sufficient viable cells for a reliable analysis. Two studies demonstrated that sputum samples collected by non-invasive methods, with the help of an assist device, contain enough viable cells that can be utilized in clinical diagnosis or research studies16,24. However, neither of these studies addressed the issue of cell numbers concerning flow cytometry.
For the studies that form the basis for this protocol, sputum samples were collected from participants at high risk for developing lung cancer following approved institutional guidelines for each study site. High-risk participants were defined as between 55-75 years, having smoked 30 pack-years and having not quit smoking within the past 15 years. Patients were shown how to use the acapella device according to the manufacturer&#39;s instructions29 and collected sputum for three consecutive days at home. The sample was kept in the refrigerator until the last collection. On the final collection day, the sample was shipped to the laboratory overnight with a frozen cold pack. The samples were processed into a single cell suspension on the day they were received. With this method of sputum collection, more than enough viable cells are obtained for a reliable flow cytometric analysis.
Lastly, and related to the previous cell number issue, is the question of how to release the sputum cells from its mucinous environment. How can the cells be kept viable and create a single cell suspension that does not clog the flow cytometer? Based on initial work by Pizzichini et al.30 and Miller et al.31, this protocol describes an easy and reliable method for sputum processing into a single cell suspension that is suitable for flow cytometric analysis. This method has used well-established guidelines in flow cytometry32,33,34 to develop an efficient antibody labeling strategy to identify hematopoietic and epithelial cells in sputum and provide instrument settings, quality control measures, and analysis guidelines standardizing sputum analysis on a flow cytometric platform.

<table><tbody><tr><td>1% Paraformaldehyde Flow-Fix</td><td>Polysciences</td><td>25037</td><td></td></tr><tr><td>100 &amp;micro;M nylon cell strainers, Falcon #352360</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>3 M NaOH</td><td>EMD</td><td>SX0593-1</td><td></td></tr><tr><td>50 mL conical falcon tube</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Alexa488 anti-human CD19</td><td>BioLegend</td><td>302219</td><td></td></tr><tr><td>Alexa488 anti-human CD3</td><td>BioLegend</td><td>300415</td><td></td></tr><tr><td>Alexa488 anti-human cytokeratin</td><td>BioLegend</td><td>628608</td><td></td></tr><tr><td>Alexa488 PanCK, CD3, and CD19 Isotype</td><td>BioLegend</td><td>400129</td><td></td></tr><tr><td>BV510 anti-human CD45</td><td>BioLegend</td><td>304036</td><td></td></tr><tr><td>CD66b FITC isotype</td><td>BD Biosciences</td><td>555748</td><td></td></tr><tr><td>CompBead Plus Compensation Beads</td><td>BD Biosciences</td><td>560497</td><td></td></tr><tr><td>Corning Polystyrene dispoable sterile bottle 250 mL</td><td>Fisher Scientific</td><td>09-761-4</td><td></td></tr><tr><td>Corning Polystyrene dispoable sterile bottle 500 mL</td><td>Fisher Scientific</td><td>09-761-10</td><td></td></tr><tr><td>CS&amp;amp;T beads</td><td>BD Biosciences</td><td>655051</td><td></td></tr><tr><td>DTT</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>FITC anti-human CD66b</td><td>GeneTex</td><td>GTX75907</td><td></td></tr><tr><td>Fixable Viability Stain</td><td>BD Biosciences</td><td>564406</td><td></td></tr><tr><td>FlowCheck</td><td>Beckman Coulter</td><td>A69183</td><td></td></tr><tr><td>FlowSet</td><td>Beckman Coulter</td><td>A69184</td><td></td></tr><tr><td>HBSS</td><td>Fisher Scientific</td><td>14-175-095</td><td></td></tr><tr><td>NAC</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>NIST Beads, 05 &amp;mu;M</td><td>Polysciences</td><td>64080</td><td></td></tr><tr><td>NIST Beads, 20 &amp;mu;M</td><td>Polysciences</td><td>64160</td><td></td></tr><tr><td>NIST Beads, 30 &amp;mu;M</td><td>Polysciences</td><td>64170</td><td></td></tr><tr><td>PE anti-human CD45</td><td>BioLegend</td><td>304039</td><td></td></tr><tr><td>PE-CF594 anti-human EpCAM</td><td>BD Biosciences</td><td>565399</td><td></td></tr><tr><td>PE-CF594 CD206/EpCAM Isotype</td><td>BD Biosciences</td><td>562292</td><td></td></tr><tr><td>PE-CR594 anti-human CD206</td><td>BD Biosciences</td><td>564063</td><td></td></tr><tr><td>Sodium citrate dihydrate</td><td>EMD</td><td>SX0445-1</td><td></td></tr><tr><td>Trypan Blue solution, 0.4%</td><td>Fisher Scientific</td><td>15250061</td><td></td></tr></tbody></table>

quality-controlled sputum analysis by flow cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

Quality-Controlled Sputum Analysis by Flow Cytometry

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

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5.0K Views.  bioAffinity Technologies. This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms. 

Video: Quality-Controlled Sputum Analysis by Flow Cytometry

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Immunology and Infection

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Bioengineering

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Biology

Identification and Enumeration of Immune Cells in Bronchoalveolar Lavage Fluid by Flow Cytometry

Source: Van Hoecke, L. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55398">Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates the use of flow cytometry to identify and count various immune cells in bronchoalveolar lavage fluid. Initially, single immune cells are identified using a viability dye, followed by differential staining of surface marker proteins, enabling the precise enumeration of each cell type.

Identyfikacja i liczenie komórek odpornościowych w płynie płucnym oskrzelowo-pęcherzykowym metodą cytometrii przepływowej

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells

Source: Flor, A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/64176">Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry.</a> J. Vis. Exp. (2022)
This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a &#946;-galactosidase substrate. The substrate is cleaved by active lysosomal &#946;-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.

Kwantyfikacja starzejących się komórek nowotworowych wywołanych terapią za pomocą cytometrii przepływowej

1. Induction of senescence by chemotherapy drugs in cultured cancer cells
NOTE: All cell manipulation steps in this section should be performed in a ...

Quality-Controlled Sputum Analysis by Flow Cytometry

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Rozdziały w tym wideo

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

Methodology for Sputum Induction and Laboratory Processing

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Identyfikacja i liczenie komórek odpornościowych w płynie płucnym oskrzelowo-pęcherzykowym metodą cytometrii przepływowej

Kwantyfikacja starzejących się komórek nowotworowych wywołanych terapią za pomocą cytometrii przepływowej