LK acknowledges the support of an Australian Government Research Training Program Scholarship.

The protocols described here are in alignment with the Declaration of Helsinki and were approved by the Ethics Committees of the German Sports University Cologne (9/16/2013) and Griffith University (2019/808). Volunteers were screened to ensure the absence of relevant pathologies and provided written informed consent.
1. Staining of RBC proteins using immunohistochemistry protocols
NOTE: A detailed list of the required chemicals and materials is provided in the Table of Materials. The following sections describe the preparation of the required solutions, followed by a detailed description of the immunohistochemistry protocol (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64843/64843fig01.jpg" /> 
Figure 1: Schematic of the individual steps required for the immunohistochemical and immunofluorescence staining of RBC-NOS at phosphorylation site 1177. A typical workflow of the presented protocols stretching from solution preparation and blood sampling to antibody-based detection and visualization is presented. <a href="https://www.jove.com/files/ftp_upload/64843/64843fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Collection of blood sample
	<ol>
		<li>Obtain blood samples (10 mL of whole blood) from seven healthy males. The volunteers were healthy individuals reportedly free from cardiovascular, hematological, neurological, endocrine, or metabolic diseases. The volunteers were also non-smokers.</li>
		<li>Collect blood using a sterile needle and syringe from a prominent vein in the antecubital region of the forearm, and immediately transfer into tubes coated with one of the following anticoagulants: sodium heparin (for immunohistochemistry) or ethylenediaminetetraacetic acid (EDTA; for fluorescent labeling).</li>
		<li>Expose the anticoagulated blood samples to precisely-controlled mechanical forces using a Couette-type shearing system, as described before7,20.
		<ol>
			<li>The shearing apparatus comprises a rotatable cup and a stationary bob, separated by a 300 &#181;m gap. Transfer the blood sample into the gap, which causes the precisely controllable rotational velocity of the cup to exert well-controlled mechanical forces onto the sample. Representative data presented here were produced by applying mechanical forces reflective of those occurring within the human vasculature (5 Pa) for extended durations (300 s).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of solutions necessary for immunostaining
	<ol>
		<li>Prepare the solutions as in Table 1. The solutions can be prepared prior to performing the immunostaining procedure and stored at given temperature until needed. 
		NOTE: Storage duration may vary between the chemicals. Check the material safety data sheet.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Process whole blood immediately after withdrawal or experimental treatment (where applicable; e.g., mechanical stimulus in our samples; see step 1.1.3) to be able to detect short-living effects, for example after shear stress application, exercise, short-duration hypoxia exposure, etc.</li>
		<li>Perform RBC protein fixation using formaldehyde as follows: dilute whole blood at a ratio of 1:2 in 4% paraformaldehyde solution for 20 min at room temperature (RT). Centrifuge the sample at 132 x g for 3 min at RT and carefully remove the supernatant by pipetting.</li>
		<li>Re-suspend the RBC pellet in two volumes of 0.1 mol/L phosphate buffered saline (PBS) (1:3 dilution) and incubate for 5 min at RT. Repeat the centrifugation as described above and remove the supernatant, which should be clear, by pipetting. Re-suspend the RBC pellet in one volume of 0.1 mol/L PBS (1:2 dilution).</li>
		<li>Prepare blood smears as follows: label a microscope slide with the sample ID, antibody applied, etc. using an alcohol resistant pen (e.g., pencil). Then, add 10 &#181;L of the prepared RBC solution right above the label field.</li>
		<li>Place a second slide on the sample at an angle of approximately 45&#176; and disperse the sample evenly along the slide. Heat-fix the slide over a Bunsen burner by hovering the sample over the burner with constant movement for 5-7 s. 
		NOTE: It is recommended to air-dry the samples prior to heat fixing. The samples can be stored at RT until staining (Figure 2).</li>
	</ol>
	</li>
	<li>Immunohistochemistry staining
	<ol>
		<li>Mark two areas on each slide: a test area in which the RBCs are incubated with the respective primary and secondary antibody, and a control area in which the primary antibody is replaced by a control solution. This area serves as a within assay control to determine the background signal of the RBCs.</li>
		<li>Prepare solutions prior to or during the procedure, as per Table 2. A volume of 300 &#181;L is needed per test area and 200 &#181;L per control area. 
		&#8203;NOTE: It is important to note that these antibody-based staining procedures yield semi-quantitative, rather than quantitative, data. Thus, to ensure that meaningful conclusions may be drawn from the produced data, appropriate control samples (i.e., to provide a reference point for the assessed relative changes in protein activation) are absolutely required.</li>
		<li>For trypsin digestion, thaw 0.1% trypsin and equilibrate to RT. Mark two areas on each slide using a grease pencil: a test area (2/3 of the slide) and a control area (1/3 of the slide). Carefully apply tris-buffered saline (TBS) using transfer pipettes to wash the sample areas. Let the TBS sit for 30 s and then pour it off. Repeat the washing step. 
		NOTE: Add the following solutions to both the control and the test area, unless otherwise described. The areas need to be sufficiently covered with the respective solution. Apply the solutions using disposable transfer pipettes, and switch pipettes after each solution.</li>
		<li>Add 0.1% trypsin, cover the slides using a purpose-built incubation chamber with either a lid or aluminum foil, and incubate for 30 min at 37 &#176;C in an incubator. Following incubation, stop the enzyme reaction by adding tap water. Pour off the solution. Wash both areas 3x&#160;with TBS, as described above.</li>
		<li>For blocking peroxidase activation, add methanol solution and incubate for 30 min at RT. Cover the slides during that time. Pour off the solution. 
		NOTE: For immunofluorescence detection (section 2), this step may be omitted, given that the blocking of endogenous peroxidases serves to prevent artifactual signal caused by the detection with horseradish peroxidase (HRP) and 3,3&#8242;-Diaminobenzidine hydrate (DAB).</li>
		<li>Wash both areas 3x with TBS, as described in step 1.4.3. Add 3% skim milk solution and incubate for 30 min at RT to block. Pour off the solution. Do not wash afterwards.</li>
		<li>Add primary antibody (AB) only to the test area. Add AB control solution (see Table 2) to the control area. Incubate samples at 4 &#176;C overnight. Cover slides during that time to prevent drying. 
		NOTE: Avoid transferring the solutions from the test to the control area, as this will influence the control values.</li>
		<li>Pour off the antibody solution. Wash both areas 3x&#160;with TBS, as described in step 1.4.3.</li>
		<li>Perform a blocking step to prevent unspecific binding. Add 3% normal goat serum&#160;and incubate for 30 min at RT. Pour off the solution.</li>
		<li>Add the secondary antibody solution and incubate for 30 min at RT. Pour off the antibody solution. Wash the areas 3x&#160;with TBS, as described in step 1.4.3.</li>
	</ol>
	</li>
	<li>Development of staining and cover
	<ol>
		<li>Carry out the avidin-coupled HRP reaction (see dilution in Table 2) by adding diluted avidin-peroxidase solution and incubating for 30 min at RT. Pour off the solution.</li>
		<li>Prepare the DAB mixture to develop the immunostaining prior to use, as per Table 3. 
		CAUTION: DAB is dangerous. Consider hazard statements H341 (suspected of causing genetic defects) and H350 (may cause cancer). Consider the following precautionary statements: P201, P202, P280, P308+P313, P405, and P501.</li>
		<li>Perform DAB control staining as follows: collect the HRP solution from step 1.5.1 from a slide and mix with a small volume (e.g., 1 mL) of prepared DAB solution in a separate centrifuge tube before staining. The mixture should develop a brown/gray color.</li>
		<li>Place the slides under a microscope (magnification of at least 200x) and add DAB solution to both areas. Monitor the staining of the RBCs continuously and stop the staining by removing the DAB solution with a disposable pipette before the background starts to color. For RBC-NOS serine 1177 staining, the DAB incubation time is about 17 min.</li>
		<li>Perform sample dehydration. Place the slides in a glass rack and immerse for 5 s each in ethanol solutions of various dilutions, starting with 70%, followed by 96%, and then 100%, and finally in xylol. Remove the slides from the rack and place on a tissue, with RBCs at the top to absorb excess liquid.</li>
		<li>Add two or three drops of mounting medium throughout the slide. Cover the slide using a coverslip. Avoid the inclusion of air bubbles, as this hinders the microscopic evaluation. Dry the samples at least overnight in a fume hood.</li>
	</ol>
	</li>
	<li>Performing microscopic evaluation
	<ol>
		<li>For visualization and imaging, place slides in a transmitted-light microscope with a magnification of at least 200x. Ensure that the microscope is coupled to a camera to take pictures of the stained RBCs.</li>
		<li>Turn on the microscope light source. Turn on the microscope-attached camera and start the microscope control software. Use brightfield microscopy to determine the appropriate focus by turning the coarse and fine focus adjustment knobs and finding the level of focus where RBCs are visible.</li>
		<li>Set the background values of the pictures to 220 &#177; 5 gray values, measured on three cell-free areas of the slide. To do so, open the first picture using the freely available software ImageJ. Select the option Mean gray values using the panel Set measurements. Use the oval icon to measure the gray value using the Measure command. If the measured values are out of range, adjust the background light at the microscope accordingly. Repeat these steps until the background values are correct. 
		NOTE: These background values should be within the given range for all images taken. Otherwise, the data are not comparable.</li>
		<li>For analysis of RBC gray value, mark the edge of each RBC using the Oval selection tool within the ImageJ software. Determine the gray values of individual RBCs using the Measure command. Measure the gray values of a minimum of 50 RBCs from the test and a minimum of 10 RBCs from the control area. 
		NOTE: The total number of analyzed RBCs can be adjusted individually. The more RBCs analyzed, the more meaningful the result. However, the total number of analyzed RBCs should be comparable for each condition, subject, etc. within a given experiment.
		<ol>
			<li>Avoid analysis of RBCs near the grease pencil, as staining might be incomplete in this area. Avoid analysis of overlapping RBCs, as this affects the staining intensity. Only use RBCs that are separated from other RBCs. Analyze at least five images from the test and at least two images from the control area for the evaluation of 50/10 RBCs. Include RBCs from every picture in the final analysis.</li>
		</ol>
		</li>
		<li>To calculate staining intensity, compute the final signals as: 
		(individual test area RBC - mean test area background) - (individual control area RBC - mean control area background)</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64843/64843fig02.jpg" /> 
Figure 2: Depiction of the fixative process and blood smear generation. (Scheme created with BioRender.com.) Diluted blood samples are chemically fixed in paraformaldehyde, then centrifuged and washed with phosphate-buffered saline. Finally, resuspended blood is smeared onto a glass slide and thermally fixed via hovering over a Bunsen burner flame. <a href="https://www.jove.com/files/ftp_upload/64843/64843fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Preparation and storage conditions for solutions necessary for immunohistochemical staining. The solution can be prepared prior to the protocol. <a href="https://www.jove.com/files/ftp_upload/64843/Table 1_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Description of antibody solutions for immediate use. <a href="https://www.jove.com/files/ftp_upload/64843/Table 2_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Components and protocol to prepare DAB solution for immediate use. <a href="https://www.jove.com/files/ftp_upload/64843/Table 3_RE.xlsx" target="_blank">Please click here to download this Table.</a>
2. Fluorescent labelling of RBC proteins
NOTE: The following section outlines an adaptation of the immunohistochemical protocol, developed with the aim of enabling the use of antibodies with fluorescent conjugates (Figure 1).
Blood sample preparation for the immunofluorescence protocol is identical to that described in section 1, so the following section commences from the staining of samples.
<ol>
	<li>Immunofluorescence staining
	<ol>
		<li>Incubate with the secondary, fluorescent conjugated antibody (for the volume, see step 1.4.2) for 30 min at RT protected from light, using either a purpose-built incubation chamber or aluminum foil.</li>
		<li>Pour off the secondary antibody solution and wash the samples 3x with TBS. Leave the final wash on the samples to prevent them from drying out.</li>
		<li>Pour off the TBS and remove the samples from the sample rack one at a time to prevent prolonged light exposure. For dehydrating the samples, expose the slides to different ethanol solutions for ~5 s each, starting with 70%, followed by 90%, and finally 100%. Expose the slides to xylol/xylene solution for 5 s.</li>
		<li>Prepare a coverslip with two or three drops of mounting medium and then mount the slide with the coverslip. Ensure even distribution of the mounting medium by applying light pressure with tweezers or a similar, sterile metal instrument. Eliminate air bubbles, which may otherwise interfere with imaging, with the same instrument. Leave the samples to dry overnight in a dark and dry space.</li>
	</ol>
	</li>
	<li>Microscopic evaluation
	<ol>
		<li>For visualization and imaging, place the slide on the microscope stage with a total magnification of at least 400x. Turn on the microscope light source and the fluorescent light source, ensuring that the fluorescent light source is adjusted to maximal intensity.</li>
		<li>Turn on the microscope-attached camera and start the microscope control software. Use brightfield microscopy to determine the appropriate focus by turning the coarse and fine focus adjustment knobs, and finding the level of focus where RBCs are visible. 
		NOTE: Excessive exposure to light may cause photobleaching of the fluorescent conjugates. One may use a slide from a previous experiment for this purpose, to minimize light exposure of the slides that are yet to be analyzed.</li>
		<li>Determine optimal laser intensity by inspecting RBCs in the control area. Ensure that the intensity is high enough that RBCs are visible, while not producing a large background signal. Keep the intensity and exposure time consistent between areas and samples to ensure comparability.</li>
		<li>Capture brightfield and fluorescent images in at least three distinct areas of the test area of the slide, selected at random. To select an area, pan away from the edges of the slide that are marked by the lipid pen, using the microscope stage controls. Select an area that exhibits uniform distribution of a singular RBC layer.</li>
		<li>Set the exposure time to 1 s for fluorescent images and capture an image using the software controls of the microscope control software. Switch to brightfield mode, set the exposure time to &#39;Auto&#39;, and capture the corresponding brightfield image.</li>
		<li>Capture brightfield and fluorescent images in at least two distinct randomly selected areas of the control area of the slide by repeating step 2.2.3.</li>
		<li>Save the images in .tif format to preserve the original gray values of pixels, prevent compression, and carry metadata of the acquisition.</li>
		<li>Measure the gray values of the captured RBCs. Open ImageJ (or FIJI21; see Supplementary File 1). Determine the gray values of individual RBCs. To this end, mark each individual cell with the Oval selection tool, and analyze using the Measure command. 
		NOTE: RBCs should not be analyzed if they overlap with other cells, as this may increase the resultant signal.</li>
		<li>Highlight between three and five areas free of RBCs and determine the gray values to provide a measure of background signal. Analyze at least 150 RBCs from at least three distinct images for each test area, and at least 50 RBCs from at least two distinct images for each control area. 
		NOTE: Analysis of more cells/areas may be advised to minimize variability. Given that the RBC population is inherently heterogenous, cell-cell variability in signal may be significant, depending on the protein of interest.</li>
		<li>Perform alternative data analysis of captured images using macro command. Create/install a macro command through the FIJI release of ImageJ for automated selection, background correction, and gray value analysis of a given image. 
		NOTE: This routine uses automated thresholding to detect the cells present in a given image using a copy of the original image, producing an overlay that is imposed onto the original image to extract gray values of fluorescent RBCs. The macro is deposited as a .ijm file, as Supplementary Coding File 1.</li>
		<li>Open the image file in .tif format ready for analysis in FIJI. Open the RBC fluorescence.ijm (Supplementary Coding File 1) macro and click Run. 
		NOTE: The macro is set up for images obtained with 600x magnification and a large signal-to-noise ratio. Automatic selection of cells should be reviewed by the investigator.</li>
	</ol>
	</li>
	<li>Compute the final signals as: 
	(test area RBC - test area background) - (control area RBC - control area background) for manual analysis; 
	(test area - control area) for automated analysis.</li>
</ol>

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<li>McMahon, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Red+blood+cell+deformability%2C+vasoactive+mediators%2C+and+adhesion%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">Red blood cell deformability, vasoactive mediators, and adhesion.</a> Frontiers in Physiology. 10, 1417(2019).</li>
<li>Bizjak, D. A., Brinkmann, C., Bloch, W., Grau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increase+in+red+blood+cell-nitric+oxide+synthase+dependent+nitric+oxide+production+during+red+blood+cell+aging+in+health+and+disease%3A+a+study+on+age+dependent+changes+of+rheologic+and+enzymatic+properties+in+red+blood+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Increase in red blood cell-nitric oxide synthase dependent nitric oxide production during red blood cell aging in health and disease: a study on age dependent changes of rheologic and enzymatic properties in red blood cells.</a> PLoS One. 10 (4), 0125206(2015).</li>
<li>Di Pietro, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitric+oxide+synthetic+pathway+and+cGMP+levels+are+altered+in+red+blood+cells+from+end-stage+renal+disease+patients%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biochemistry%22%5BJournal%5D)">Nitric oxide synthetic pathway and cGMP levels are altered in red blood cells from end-stage renal disease patients.</a> Molecular and Cellular Biochemistry. 417 (1-2), 155-167 (2016).</li>
<li>Grau, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Even+patients+with+mild+COVID-19+symptoms+after+SARS-CoV-2+infection+show+prolonged+altered+red+blood+cell+morphology+and+rheological+parameters%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">Even patients with mild COVID-19 symptoms after SARS-CoV-2 infection show prolonged altered red blood cell morphology and rheological parameters.</a> Journal of Cellular and Molecular Medicine. 26 (10), 3022-3030 (2022).</li>
<li>Mozar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Red+blood+cell+nitric+oxide+synthase+modulates+red+blood+cell+deformability+in+sickle+cell+anemia%5BTitle%5D)+AND+%22Clinical+Hemorheology+and+Microcirculation%22%5BJournal%5D)">Red blood cell nitric oxide synthase modulates red blood cell deformability in sickle cell anemia.</a> Clinical Hemorheology and Microcirculation. 64 (1), 47-53 (2016).</li>
<li>Ulker, P., Gunduz, F., Meiselman, H. J., Baskurt, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitric+oxide+generated+by+red+blood+cells+following+exposure+to+shear+stress+dilates+isolated+small+mesenteric+arteries+under+hypoxic+conditions%5BTitle%5D)+AND+%22Clinical+Hemorheology+and+Microcirculation%22%5BJournal%5D)">Nitric oxide generated by red blood cells following exposure to shear stress dilates isolated small mesenteric arteries under hypoxic conditions.</a> Clinical Hemorheology and Microcirculation. 54 (4), 357-369 (2013).</li>
<li>Nader, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydroxyurea+therapy+modulates+sickle+cell+anemia+red+blood+cell+physiology%3A+Impact+on+RBC+deformability%2C+oxidative+stress%2C+nitrite+levels+and+nitric+oxide+synthase+signalling+pathway%5BTitle%5D)+AND+%22Nitric+Oxide%3A+Biology+and+Chemistry%22%5BJournal%5D)">Hydroxyurea therapy modulates sickle cell anemia red blood cell physiology: Impact on RBC deformability, oxidative stress, nitrite levels and nitric oxide synthase signalling pathway.</a> Nitric Oxide: Biology and Chemistry. 81, 28-35 (2018).</li>
<li>Fischer, U. M., Schindler, R., Brixius, K., Mehlhorn, U., Bloch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracorporeal+circulation+activates+endothelial+nitric+oxide+synthase+in+erythrocytes%5BTitle%5D)+AND+%22The+Annals+of+Thoracic+Surgery%22%5BJournal%5D)">Extracorporeal circulation activates endothelial nitric oxide synthase in erythrocytes.</a> The Annals of Thoracic Surgery. 84 (6), 2000-2003 (2007).</li>
<li>Horobin, J. T., Sabapathy, S., Kuck, L., Simmonds, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shear+stress+and+RBC-NOS+Serine1177+Phosphorylation+in+humans%3A+a+dose+response%5BTitle%5D)+AND+%22Life%22%5BJournal%5D)">Shear stress and RBC-NOS Serine1177 Phosphorylation in humans: a dose response.</a> Life. 11 (1), 36(2021).</li>
<li>Kuck, L., Grau, M., Simmonds, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recovery+time+course+of+erythrocyte+deformability+following+exposure+to+shear+is+dependent+upon+conditioning+shear+stress%5BTitle%5D)+AND+%22Biorheology%22%5BJournal%5D)">Recovery time course of erythrocyte deformability following exposure to shear is dependent upon conditioning shear stress.</a> Biorheology. 54 (5-6), 141-152 (2018).</li>
<li>Grau, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+acute+exercise+on+RBC+deformability+and+RBC+nitric+oxide+synthase+signalling+pathway+in+young+sickle+cell+anaemia+patients%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Effect of acute exercise on RBC deformability and RBC nitric oxide synthase signalling pathway in young sickle cell anaemia patients.</a> Scientific Reports. 9 (1), 11813(2019).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Methods+in+Nitric+Oxide+Research%22">Methods in Nitric Oxide Research</a>. Feelisch, M. , Wiley. Chichester. (1998).</li>
<li>Cortese-Krott, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+red+blood+cells+at+work%3A+identification+and+visualization+of+erythrocytic+eNOS+activity+in+health+and+disease%5BTitle%5D)+AND+%22Blood%3E%22%5BJournal%5D)">Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease.</a> Blood>. 120 (20), 4229-4237 (2012).</li>
</ol>

All authors have disclosed that there are no conflicts of interest.

Recent literature highly suggests that the RBC-NOS protein is of crucial importance for the regulation of RBC deformability15,22,23, which in turn facilitates their passage through narrow capillaries24. Protein activity highly depends on post-translational protein modifications, particularly the phosphorylation of certain residues18. The focus of interest lies in the phosphorylatio...

Red blood cells (RBCs) traverse the cardiovascular system for 70 to 140 days, with a mean RBC age of approximately 115 days1,2. Senescent or damaged RBCs are removed from the circulation by erythrophagocytosis, an efficient clearing process driven by macrophages3. The predetermined lifespan of these cells is one consequence of surrendering the cell organelles, including the nucleus, mitochondria, and ribosomes, during differentiation and maturation4. Thus, circulating RBCs are devoid of a translational machinery, precluding the synthesis of new proteins3. It follows that dynamic, post-translational modifications to existing proteins represent the only viable mechanism of acute, biochemical regulation in response to extracellular and intracellular stressors acting on RBCs5.
Mechanical forces appear to be chief extracellular cues that cause the activation or modulation of biochemical pathways within RBCs. The discovery of the mechanosensitive protein, Piezo1, in RBC membranes6 inspired several lines of research investigating mechanically-activated signaling in these cells7. For example, recent advances have shown that the physical properties of RBCs are actively regulated by acute and dynamic changes of proteins8, which includes post-translational phosphorylation and ubiquitination9. Since these normal modifications differ in certain diseases9,10,11, it seems to be of scientific and clinical interest to determine the activation state of RBC proteins, specifically in relation to mechanobiological processes.
The determination of acute changes in RBC protein activation states poses some methodological challenges. For instance, the storage of RBC samples for later analysis requires preservation of the modified RBC proteins, as post-translational modifications are non-durable. Moreover, classic protein-detection methods (e.g., western blotting) are notoriously difficult to standardize in RBCs due to the low abundance of proteins relative to hemoglobin, which accounts for ~98% of the protein content in these cells12. Thus, antibody-based staining of chemically-preserved RBCs has been the method of choice when investigating acute modifications of important RBC proteins, such as the RBC-specific isoform of nitric oxide synthase (RBC-NOS)13,14. RBC-NOS has been shown to enzymatically produce nitric oxide (NO), which seems indispensable for essential RBC properties, including RBC deformability15,16,17. Post-translational modifications of RBC-NOS regulate catalytic enzyme activity, with phosphorylation of the serine 1177 residue being described to increase enzyme activity, while phosphorylation of the residues serine 114 or threonine 495 have been linked with decreased RBC-NOS activity18,19.
Collectively, temporary modifications of RBC proteins contribute to important cellular function, and standardized protocols that enable detection of these modified proteins are of high value. Here, we present two distinct protocols that exploit specific antibodies to facilitate the detection of RBC-NOS protein activation, and discuss recommendations for data analysis and interpretation.
Performance of the described protocols was assessed by measuring the well-reported increase in the phosphorylation of RBC-NOS at the serine 1177 residue in response to mechanical forces reflective of those occurring within the human vasculature (5 Pa).

<table><tbody><tr><td>3,3&amp;prime;-Diaminobenzidin -tetrahydrochloride Hydrate</td><td>Sigma/Merck</td><td>D5637</td><td>DAB</td></tr><tr><td>Ammoniumchloride&amp;nbsp;</td><td>Merck /Millipore</td><td>101145</td><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td></tr><tr><td>Centrifuge 5427 R&amp;nbsp;</td><td>Eppendorf</td><td>5409000010</td><td></td></tr><tr><td>Coverslips</td><td>VWR</td><td>631-0147&amp;nbsp;</td><td></td></tr><tr><td>di-sodium Hydrogen Phosphate Dihydrate&amp;nbsp;</td><td>Merck /Millipore</td><td>106580</td><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;. 2 H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Disposable transfer pipettes</td><td>VWR</td><td>612-6803</td><td></td></tr><tr><td>Entellan</td><td>Merck /Millipore</td><td>107961</td><td>rapid mounting medium for microscopy</td></tr><tr><td>Ethanol denaturated using 1 % methyl ethyl ketone (MEK)</td><td>Hofmann</td><td>642</td><td></td></tr><tr><td>Glucose-Oxidase</td><td>Sigma/Merck</td><td>G2133</td><td></td></tr><tr><td>Grease pencil&amp;nbsp;</td><td>Dako</td><td>S 2002</td><td></td></tr><tr><td>Horse-radish peroxidase/ExtrAvidin&amp;minus;Peroxidase</td><td>Sigma/Merck</td><td>E-2886</td><td>HRP</td></tr><tr><td>Hydrochloric acid&amp;nbsp;</td><td>Merck /Millipore</td><td>109057</td><td>HCl</td></tr><tr><td>Hydrogen peroxide, 30%</td><td>Merck /Millipore</td><td>107203</td><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>ImageJ Software</td><td>Freeware</td><td></td><td></td></tr><tr><td>Laser-assisted optical rotational cell analyser (LORCA)</td><td>RR Mechatronics</td><td></td><td>Ektacytometer instrument used for shearing</td></tr><tr><td>Methanol</td><td>Merck /Millipore</td><td>106009</td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>630-1985</td><td></td></tr><tr><td>Nickel(II)-sulfate Hexahydrate&amp;nbsp;</td><td>Sigma/Merck</td><td>N4882</td><td>NiSO&lt;sub&gt;4.&lt;/sub&gt;6H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Normal Goat serum</td><td>Agilent/DAKO</td><td>X0907</td><td>NGS</td></tr><tr><td>Paraformaldehyde</td><td>Merck /Millipore</td><td>818715</td><td>PFA</td></tr><tr><td>Pipettes Eppendorf Reference 2</td><td>VWR</td><td>613-5836/ 613-5839</td><td></td></tr><tr><td>Rabbit Anti-phospho eNOS Antibody (Ser1177)</td><td>Merck/Millipore</td><td>07-428-I</td><td>Primary Antibody</td></tr><tr><td>Reaction tubes, 2ml</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>Secondary Antibody goat anti rabbit</td><td>Agilent/DAKO</td><td>E0432</td><td>Secondary Antibody</td></tr><tr><td>Skim milk powder</td><td>Bio-Rad</td><td>170-6404</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Merck /Millipore</td><td>106404</td><td>NaCl</td></tr><tr><td>Sodium Dihydrogen Phosphate Monohydrate</td><td>Merck /Millipore</td><td>106346</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sodium hydroxide, 1 M</td><td>Merck /Millipore</td><td>150706</td><td>NaOH</td></tr><tr><td>Tris(hydroxymethyl)-aminomethane</td><td>Merck /Millipore</td><td>108382</td><td>Tris</td></tr><tr><td>Trypsin</td><td>Sigma/Merck</td><td>T7409</td><td></td></tr><tr><td>Tween20&amp;nbsp;</td><td>Merck /Millipore</td><td>822184</td><td></td></tr><tr><td>Whatman Glas microfiber filter, quality GF/F</td><td>Merck /Millipore</td><td>WHA1825047</td><td></td></tr><tr><td>Xylol</td><td>VWR Chemicals</td><td>2,89,73,465</td><td></td></tr><tr><td>&amp;szlig;-D-Glucose monohydrate</td><td>Merck /Millipore</td><td>14431-43-7</td><td></td></tr></tbody></table>

immunostaining-based detection of dynamic alterations in red blood cell proteins

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

The presented protocol, describing methods that facilitate the detection of acute alterations in RBC proteins, was tested on a well-known mechanically sensitive protein alteration: phosphorylation of RBC-NOS at the serine 1177 residue. Whole blood was obtained from healthy volunteers and subsequently split into two separate aliquots. A given blood sample was exposed to mechanical shear stress of physiological magnitude (5 Pa) for 300 s, which was previously shown to elicit RBC-NOS phosphorylation at serine 1177<sup class...

Watch this Scientific Journal Video about Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins at JoVE.com

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Capturing dynamic changes in the protein activation of enucleated red blood cells poses methodological challenges, like the preservation of dynamic changes to acute stimuli for later assessment. The presented protocol describes sample preparation and staining techniques that enable preservation and analysis of relevant protein changes and subsequent detection.

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute ...

immunostaining-based-detection-dynamic-alterations-red-blood-cell

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

1.9K Views.  German Sport University Cologne. Capturing dynamic changes in the protein activation of enucleated red blood cells poses methodological challenges, like the preservation of dynamic changes to acute stimuli for later assessment. The presented protocol describes sample preparation and staining techniques that enable preservation and analysis of relevant protein changes and subsequent detection.

Video: Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Developmental Biology

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Analysis Workflow of Nuclear Protein Staining in Human Primary Cells

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience.
In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution.
Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different&#160;contexts.

Author Spotlight: Comprehensive Epigenetic Analysis for Investigating Human Cellular Plasticity and Environmental Adaptation Using Immunofluorescence Assays

This method describes an immunofluorescence protocol and quantification pipeline for evaluating protein distribution with varied nuclear organization patterns in human T lymphocytes. This protocol provides step-by-step guidance, starting from sample preparation and continuing through the execution of semi-automated analysis in Fiji, concluding with data handling by a Google Colab notebook.

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the ...

Genetics

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Medicine

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Biology

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Summary

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Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells