Alternative Lymphatic Drainage Pathways in the Trunk Following Oncologic Therapy.

BACKGROUND: Anatomic and functional descriptions of trunk and breast lymphedema following breast cancer treatment are emerging as indicators of lymphatic dysfunction. Indocyanine green-lymphangiography has been instrumental in characterizing this dysfunction in the extremity and can be applied to other regions. Previous work has established a validated Pittsburgh Trunk Lymphedema Staging System to characterize such affected areas. This study aims to identify risk and protective factors for the development of truncal and upper extremity lymphedema using alternative lymphatic drainage, providing implications for medical and surgical treatment. METHODS: Patients undergoing revisional breast surgery with suspicion of upper extremity lymphedema between 12/2014 and 3/2020 were offered lymphangiography. The breast and lateral/anterior trunks were visualized and blindly evaluated for axillary and inguinal lymphatic flow. A linear-weighted Cohen's kappa statistic was calculated comparing alternative drainage evaluation. Binomial regression was used to compute relative risks (RRs). Significance was assessed at alpha = 0.05. RESULTS: Eighty-six sides (46 patients) were included. Twelve sides underwent no treatment and were considered controls. Eighty-eight percent of the noncontrols had alternative lymphatic flow to the ipsilateral axillae (64%), ipsilateral groins (57%), contralateral axillae (20.3%), and contralateral groins (9.3%). Cohen's kappa for alternative drainage was 0.631 ± 0.043. Ipsilateral axillary and contralateral inguinal drainage were associated with reduced risk of developing truncal lymphedema [RR 0.78, confidence interval (CI) 0.63-0.97, P = 0.04; RR 0.32, CI 0.13-0.79, P = 0.01, respectively]. Radiation therapy increased risk of truncal and upper extremity lymphedema (RR 3.69, CI 0.96-14.15, P = 0.02; RR 1.92, CI 1.09-3.39, P = 0.03, respectively). Contralateral axillary drainage and axillary lymph node dissection were associated with increased risk of upper extremity lymphedema (RR 4.25, CI 1.09-16.61, P = 0.01; RR 2.83, CI 1.23-6.52, P = 0.01, respectively). CONCLUSIONS: Building upon previous work, this study shows risk and protective factors for the development of truncal and upper extremity lymphedema. Most prevalent alternative channels drain to the ipsilateral axilla and groin. Ipsilateral axillary and contralateral inguinal drainage were associated with reduced risk of truncal lymphedema. Patients with radiation, axillary dissection, and contralateral axillary drainage were associated with increased risk of upper extremity lymphedema. These findings have important clinical implications for postoperative manual lymphatic drainage and for determining eligibility for lymphovenous bypass surgery.

Nipple Areola Complex Reconstruction With the "Half-Dome" Technique Following Implant-Based Breast Reconstruction.

BACKGROUND: No single technique for nipple areola reconstruction best fits every patient and clinical scenario. Many techniques fail to provide long-term projection. One especially challenging cohort are those patients who have undergone bilateral implant-based reconstruction. We developed a modification of the C-V flap reconstruction that increases projection in the bilateral, implant-based reconstruction patient. METHODS: All patients who underwent nipple areola reconstruction following implant-based breast reconstruction and who had at least a 12-month follow-up visit were identified. Nipple projection was measured and compared between the 2 groups. RESULTS: Forty patients were identified. Twelve patients, 23 nipples, underwent the standard C-V flap reconstruction. Twenty-eight patients, 59 nipples, underwent the half-dome modification. Average nipple projection following the half-dome technique is more than twice that of the C-V flap. CONCLUSIONS: The half-dome technique provides a useful alternative modification of the C-V flap in patients with implant-based reconstruction.

Characterizing Breast Deformities After Massive Weight Loss: Utilizing the Pittsburgh Rating Scale to Examine Factors Affecting Severity Score and Surgical Decision Making in a Retrospective Series.

BACKGROUND AND OBJECTIVES: Massive weight loss (MWL) can result in variable contour deformities of the breasts. The Pittsburgh Rating Scale (PRS) was designed to describe the multitude of deformities after MWL and recommends operations to consider for surgical improvement. We present the first comprehensive description of breast deformities in a large sample of MWL patients, examine factors affecting the severity of deformities, and report the correlation between PRS score and surgical decision making. METHODS: A retrospective review of all MWL patients presenting for breast surgery at our institution's Life After Weight Loss program from 2004 to 2015 was performed. Information including demographics, body mass indices (BMIs), method of weight loss, and type of surgical intervention was collected. Preoperative breast photographs were blinded and scored according to the PRS. RESULTS: A total of 204 MWL patients were identified; 26% (53) scored 1, 34% (69) scored 2, and 40% (82) scored 3 on the PRS. Greater deformities were seen after weight loss from bariatric surgery versus diet and exercise alone (P = 0.031), in mastopexy versus augmentation/mastopexy (P = 0.001), and in breast reduction versus augmentation/mastopexy patients (P > 0.0001). Patients who underwent reduction mammaplasty had the greatest maximum BMI compared with other procedures (P = 0.016). The PRS scores were positively correlated to maximum BMI (P < 0.001), delta BMI (P < 0.001), and current BMI (P < 0.001). CONCLUSIONS: Massive weight loss patients have variable, and often severe, breast deformities, and the PRS remains a valuable classification tool. Severity scores correlate with BMI, procedure, and weight loss mechanism. Similar scores between mastopexy-only and reduction mammaplasty patients may reflect a composite of personal cosmetic expectations and cost. The PRS scale should also be expanded to include breast reduction as a surgical remedy for PRS grade 3 breast deformities. Understanding breast deformities in this unique population has applications in both preoperative planning and surgical expectations for this unique patient population.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

Intraluminal nonbacterial intestinal components control gut and lung injury after trauma hemorrhagic shock.

OBJECTIVE: To test whether the mucus layer, luminal digestive enzymes, and intestinal mast cells are critical components in the pathogenesis of trauma shock-induced gut and lung injury. BACKGROUND: Gut origin sepsis studies have highlighted the importance of the systemic component (ischemia-reperfusion) of gut injury, whereas the intraluminal component is less well studied. METHODS: In rats subjected to trauma hemorrhagic shock (T/HS) or sham shock, the role of pancreatic enzymes in gut injury was tested by diversion of pancreatic enzymes via pancreatic duct exteriorization whereas the role of the mucus layer was tested via the enteral administration of a mucus surrogate. In addition, the role of mast cells was assessed by measuring mast cell activation and the ability of pharmacologic inhibition of mast cells to abrogate gut and lung injury. Gut and mucus injury was characterized functionally, morphologically, and chemically. RESULTS: Pancreatic duct exteriorization abrogated T/HS-induced gut barrier loss and limited chemical mucus changes. The mucus surrogate prevented T/HS-induced gut and lung injury. Finally, pancreatic enzyme-induced gut and lung injury seems to involve mast cell activation because T/HS activates mast cells and pharmacologic inhibition of intestinal mast cells prevented T/HS-induced gut and lung injury. CONCLUSIONS: These results indicate that gut and gut-induced lung injury after T/HS involves a complex process consisting of intraluminal digestive enzymes, the unstirred mucus layer, and a systemic ischemic-reperfusion injury. This suggests the possibility of intraluminal therapeutic strategies.

Trauma-hemorrhagic shock induces a CD36-dependent RBC endothelial-adhesive phenotype.

OBJECTIVE: Microvascular dysfunction is a key element in the development of the multiple organ dysfunction syndrome. Although the mechanisms for this response are unclear, RBC adhesion to endothelium may initiate intravascular occlusion leading to ischemic tissue injury. Thus, we tested the hypothesis that trauma-hemorrhage induces RBC-endothelial cell adhesion. DESIGN: Prospective in vivo and in vitro animal study and analysis of patient blood samples. SETTING: University research laboratory and hospital emergency and trauma units. INTERVENTION: We initially assayed RBC adhesion to endothelial cells in vitro using RBCs obtained from rats subjected to trauma-hemorrhagic shock or sham shock as well as from severely injured trauma patients. Subsequently, we measured the role of putative RBCs and endothelial cell receptors in the increased RBC-endothelial cell adhesive response. MAIN RESULTS: In both rats and humans, trauma-hemorrhagic shock increased RBC adhesion to endothelium as well as increasing several putative RBC surface adhesion molecules including CD36. The critical factor leading to RBC-endothelial cell adhesion was increased surface RBC CD36 expression. Adhesion of trauma-hemorrhagic shock RBCs was mediated, at least in part, by the binding of RBC CD36 to its cognate endothelial receptors (αVß3 and VCAM-1). Gut-derived factors carried in the intestinal lymphatics triggered these trauma-hemorrhagic shock-induced RBC changes because 1) preventing trauma-hemorrhagic shock intestinal lymph from reaching the systemic circulation abrogated the RBC effects, 2) in vitro incubation of naïve whole blood with trauma-hemorrhagic shock lymph replicated the in vivo trauma-hemorrhagic shock-induced RBC changes while 3) injection of trauma-hemorrhagic shock lymph into naïve animals recreated the RBC changes observed after actual trauma-hemorrhagic shock. CONCLUSIONS: 1) Trauma-hemorrhagic shock induces rapid RBC adhesion to endothelial cells in patients and animals. 2) Increased RBC CD36 expression characterizes the RBC-adhesive phenotype. 3) The RBC phenotypic and functional changes were induced by gut-derived humoral factors. These novel findings may explain the microvascular dysfunction occurring after trauma-hemorrhagic shock, sepsis, and other stress states.

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens.Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and ELISA. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens.

Oxidative modification of the intestinal mucus layer is a critical but unrecognized component of trauma hemorrhagic shock-induced gut barrier failure.

Recent studies demonstrate that mechanisms underlying gut barrier failure include systemic processes and less studied luminal processes. We thus tested the hypothesis that mucus layer oxidation is a component of trauma/hemorrhagic shock-induced gut injury and dysfunction. Male Sprague-Dawley rats underwent trauma/hemorrhagic shock. Controls underwent trauma only. Mucus from the terminal 30 cm of the ileum was collected, processed, and analyzed for reactive nitrogen intermediates (RNI)-mediated damage, reactive oxygen species (ROS)-induced damage, and total antioxidant capacity. The distal ileum was stained to quantify the mucus layer; gut permeability was assessed physiologically. A time course study was conducted to determine the temporal sequence of mucus layer damage. The role of free radical-mediated damage to the gut barrier was investigated by the effect of the free radical scavenger dimethyl sulfoxide on trauma/hemorrhagic shock-induced changes on the mucus and on gut permeability. Trauma/hemorrhagic shock increased intestinal permeability, which was associated with evidence of loss of the unstirred mucus layer. These changes correlated with increased ROS- and RNI-mediated mucus damage and loss of mucus total antioxidant capacity. Based on the time course study, ROS-mediated mucus damage and loss of total antioxidant capacity were present immediately following shock, whereas RNI-mediated damage was delayed for 3 h. Dimethyl sulfoxide ameliorated gut barrier loss, ROS-mediated changes to the mucus layer, and loss of total antioxidant capacity. There was no change in RNI-induced changes to the mucus layer. These results support the hypothesis that trauma/hemorrhagic shock leads to mucus damage and gut dysfunction through the generation of free radical species.

The Pittsburgh Trunk Lymphedema Staging System (PTLSS) - a validated staging system for the description of breast cancer-associated trunk lymphedema.

BACKGROUND: Neither anatomic nor functional descriptions exist of trunk/breast lymphedema following breast cancer treatment. Indocyanine green (ICG)-lymphangiography has been shown to characterize lymph channel dysfunction seen in lymphedema. We propose using ICG-lymphangiography to evaluate trunk and breast lymphedema following breast cancer surgery to characterize the regions affected via a novel, validated staging system. METHODS: Patients undergoing revisional breast surgery with suspicion of upper extremity lymphedema between December 2014 and March 2020 were offered lymphangiography. The breast and lateral/anterior trunks were visualized and blindly evaluated using Koshima's patterns of dermal backflow. Patients were then staged. A linear-weighted Cohen's kappa statistic was calculated comparing each rated area and stage assignment. RESULTS: Fifty-two sides (29 patients) were included. Eight sides underwent no treatment and were considered controls. No lymphedema was identified within this cohort. One patient (two sides) had no transit of ICG. Seventy-six percent of the non-controls had dermal backflow. This was seen in 67% of anterior trunks, 50% of lateral trunks, 50% of inframammary folds (IMFs), 43% of inferior breasts, and 5% of superior breasts. Cohen's kappa for area agreement was 0.4117 ± 0.0535. Stage 0 was seen in 31 (±7)% of sides; stage 1: 21 (±1)%; stage 2: 22 (±5)%; stage 3: 18 (±4)%; stage 4: 5 (±1)%; and stage 5: 4 (±0). Cohen's kappa for staging was 0.8109 ± 0.0868. CONCLUSION: Following breast cancer surgery, lymphedema occurs throughout the trunk and breast. Severe dysfunction appears to be located around the inferior-lateral aspect of the breast and chest wall. Furthermore, the Pittsburgh Trunk Lymphedema Staging System is a validated measure of trunk and breast lymphedema.

Modulation of the protein kinase Cdelta interaction with the "d" subunit of F1F0-ATP synthase in neonatal cardiac myocytes: development of cell-permeable, mitochondrially targeted inhibitor and facilitator peptides.

The F(1)F(0)-ATP synthase provides approximately 90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cdelta (PKCdelta) inhibits F(1)F(0) activity via an interaction with the "d" subunit of F(1)F(0)-ATP synthase (dF(1)F(0)) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831-29840). We have now identified a dF(1)F(0)-derived peptide (NH(2)-(2)AGRKLALKTIDWVSF(16)-COOH) that inhibits PKCdelta binding to dF(1)F(0) in overlay assays. We have also identified a second dF(1)F(0)-derived peptide (NH(2)-(111)RVREYEKQLEKIKNMI(126)-COOH) that facilitates PKCdelta binding to dF(1)F(0). Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCdelta-dF(1)F(0) interaction inhibitor, decreased 100 nm 4beta-phorbol 12-myristate 13-acetate (4beta-PMA)-induced co-immunoprecipitation of PKCdelta with dF(1)F(0) by 50 +/- 15% and abolished the 30 nm 4beta-PMA-induced inhibition of F(1)F(0)-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCdelta-dF(1)F(0) facilitator peptide by itself induced the PKCdelta-dF(1)F(0) co-immunoprecipitation and inhibited F(1)F(0)-ATPase activity. In in vitro PKC add-back experiments, the PKCdelta-F(1)F(0) inhibitor blocked PKCdelta-mediated inhibition of F(1)F(0)-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCdelta-dF(1)F(0) interaction in NCMs. These novel peptides will improve our understanding of cardiac F(1)F(0) regulation and may have potential as therapeutics to attenuate cardiac injury.

Labeling Antibodies Using Europium.

There are many uses for antibodies labeled with metal ions. Most of these methods involve first attaching a metal chelator to the antibody molecule. This is achieved using standard cross-linking chemistry and then adding the desired metal at appropriate concentration and pH. The method described here outlines a basic procedure for creating a lanthanide conjugate. Lanthanide conjugates are used for proximity assays, as MRI contrast agents, or for mass cytometry experiments. Different metals and chelators can be substituted, but the basic procedures are similar.

Labeling Antibodies Using Colloidal Gold.

Colloidal gold-antibody conjugates are easy to prepare and are an excellent choice for microscopic applications. Colloidal gold is an aqueous suspension of nanometer-sized particles of gold. Typically, chloroauric acid, HAuCl4, is reduced with dilute solutions of sodium citrate, as described here. This will cause the gold to form small aggregates that will associate with proteins. Gold particles of specific sizes can be isolated and differentiated microscopically, allowing these particles to be used for multiple-label experiments. Colloidal gold-labeled antibodies are widely used in electron microscopy (EM), and can be used for light microscopy but require additional steps (silver enhancement).

Labeling Antibodies with N-Hydroxysuccinimide-Long Chain (NHS-LC)-Biotin.

Labeling antibodies with biotin (biotinylation) is a useful and simple technique. Biotin's small size (244 Da) usually has little effect on the biological activity of the protein target. The most common way to biotinylate an antibody is to cross-link a biotin succinimidyl ester to a primary amine. There are many commercially available types of biotin analogs that can be used for labeling. They vary in reactive group chemistry as well as spacer length. For example, a common analog used for biotinylation is the succinimidyl ester of biotin with an aminohexanoic acid spacer (Long Chain or LC-Biotin), utilized here. A PEG spacer of varying length can also be used.

Biotinylating Antibodies Using Biotin Polyethylene Oxide (PEO) Iodoacetamide.

There are several techniques for biotinylating antibodies, from the most basic (using NHS-ester biotin to label primary amines) to more complex experiments (modifying sulfhydryls and carbohydrates). Biotinylation of free sulfhydryls, described here, can be effectively mediated using haloacetyl biotin derivatives. To modify an antibody using this reagent, sulfhydryls must be available. Digestion of antibodies by the enzyme pepsin produces F(ab')2 fragments, which can be separated by mild reduction into two sulfhydryl-containing, univalent Fab' fragments. Alternatively, thiol groups can be added by modifying amines with an appropriate cross-linker.

Biotinylating Antibodies Using Biotin-Long Chain (LC) Hydrazide.

Hydrazide derivatives are useful for biotinylating antibodies at oxidized carbohydrate groups. This protocol uses oxidation conditions that will convert most if not all possible hydroxyl sites to aldehydes. Each antibody may require different oxidation conditions to optimize labeling. Some monoclonal antibodies may be deficient in glycosylation, making this method suboptimal. Other labeling reagents (fluorophores) are also available as hydrazides. Hetero-bifunctional cross-linkers (e.g., ß-maleimidopropionic acid hydrazide [BMPH]) are also available to cross-link other targets to carbonyls.

Iodination of Antibodies with Immobilized Iodogen.

Iodination, a chemical or enzymatic incorporation of 125I to specific amino acid side chains, is a commonly used method for labeling antibodies with radioisotopes. Commercially available products make iodination of antibodies a simple and quick process. One example, used here and available at Pierce, is the "Iodination bead," or N-chloro-benzenesulfonamide immobilized on nonporous, polystyrene beads.

Purification of Labeled Antibodies Using Size-Exclusion Chromatography.

Many antibody labeling procedures call for a desalting or purification step requiring size-exclusion chromatography (SEC). The method outlined here contains information needed to desalt an antibody conjugate. Similar procedures would be used for ion-exchange chromatography using a gradient of increasing ionic strength. Resins can be purchased in bulk (as in this protocol), or commercially available columns are available.

Labeling Antibodies.

This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific site of modification, cross-linker options, types of labels, and postlabeling cleanup methodology, along with the advantages and disadvantages of each method. In general, polyclonal antibodies are more versatile and resistant to activity loss than are monoclonal antibodies. Greater care must be taken when labeling monoclonal antibodies to ensure a quality conjugate. The methods outlined here can be adapted for a variety of labels including multiple labels on the same immunoglobulin. The most important consideration when undertaking an antibody labeling experiment is to maintain the activity of the antibody. This is an empirical process and will often require additional experiments to optimize the label of a particular antibody. When successful, these reagents are very useful and adaptable biomolecules. This introduction provides the reader with methods and options for producing a variety of labeled immunological tools.

Labeling Antibodies Using N-Hydroxysuccinimide (NHS)-Fluorescein.

N-Hydroxysuccinimide (NHS)-ester derivatives are among the most commonly used reagents for labeling proteins. The method described here can be adapted to use practically any NHS fluorophore. Generally, a fluorophore is covalently bound to a macromolecule such as an antibody and acts as a reporter molecule used to measure the presence of the macromolecule. These fluorescently labeled bioactive reagents are suitable for use in immunofluorescence, flow cytometry, and numerous other biological applications. There are several widely used dyes available in convenient formats. This protocol can be used with any amine-reactive (e.g., PFP, isothiocyanate) fluorophore derivative.