Name: A Quantitative Assessment of the Phagocytosis of Allogeneic and Xenogeneic Erythrocytes by Rat Macrophages In Vitro (Video) | JoVE Journal
Uploaded: 2025-08-22T14:05:00.000+00:00
Duration: 9 min 1 s
Description: Learn a standardized in vitro method to quantify phagocytosis of xenogeneic and allogeneic erythrocytes by rat macrophages. This protocol establishes a relative phagocytosis index for immunology research.

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5 minutes

We would like to thank Ms Mei Fang, Ms Min Ma, Ms Yu-Tong Meng, and Mr. Quan-Kai Lei for their initial contributions. This research was funded partly by the National Natural Science Foundation of China (82270697), the Science and Technology Planning Project of Guangdong Province of China (2021B1212040016), the Guangdong Basic and Applied Basic Research Foundation (2023A1515012574), the Jiangsu Provincial Medical Key Discipline Cultivation Unit (JSDW202229) and the Haihe Laboratory of Cell Ecosystem Innovation Fund (HH24KYZX0008), China Foundation For Youth Entrepreneurship and Employment -Incaier Public Welfare Fund (HH25KYHX0003).

All experiments involving animals were conducted according to the ethical policies and procedures approved by The Institutional Animal Care and Use Committee of Jiangsu University (UJS-IACUC) (Approval no. UJS-IACUC-2021090102). This study was approved by the Institutional Review Board of The Affiliated Hospital of Jiangsu University. All samples were collected from patients who had provided informed consent. The removed supernatant should be placed in a dedicated waste liquid tank and handed over to the hospital&#39;s infectious disease department.
1. Macrophage isolation
<ol>
	<li>Preparation: Prepare the necessary materials, including experimental rats, anesthesia containers, beakers, and isoflurane as the anesthetic, and pre-cool the centrifuge at 4 &#176;C.</li>
	<li>Anesthetizing and euthanizing&#160;the rats: Following the institutional animal care guidelines, anesthetize animals with inhaled isoflurane during experiments and then euthanize with CO2 post-experiment.</li>
	<li>Alcohol disinfection and incision: Disinfect the rat&#39;s abdomen with ethanol and make a small incision in the rat&#39;s abdominal wall.</li>
	<li>Injection and mixing of abdominal fluid: Inject 50 mL of ice-cold sterile PBS into the abdominal cavity along the midline using a syringe. Gently shake the rat and massage the abdominal wall with fingers to ensure thorough mixing of the fluid in the abdominal cavity. Massage for 2-3 mins.</li>
	<li>Collection of the abdominal fluid: Tilt the body slightly, and aspirate the abdominal fluid with a syringe.</li>
	<li>Centrifugation: Centrifuge the collected fluid at 500 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
	<li>RBCs Lysis: Add 3-5 times the cell pellet volume of RBC lysis buffer and gently mix for 1-2 min. 
	NOTE: For example, if the cell pellet volume is 1 mL, add 3-5 mL of lysis buffer. This step can be performed at room temperature or at 4 &#176;C, followed by centrifugation at 400-500 x g for 5 min. Centrifugation at 4 &#176;C is more effective. Due to the osmotic pressure, RBCs will lyse, and the solution will turn red. After centrifugation, remove the red supernatant.
	<ol>
		<li>Red Blood Cell (RBC) lysis buffer recipe: Dissolve 8.3 g of NH4Cl, 1.0 g of KHCO3, and 1.8 mL of 5% EDTA in 800 mL of distilled H2O, and filter sterilize through a 0.2 &#181;m filter, qs to 1000 mL with distilled H2O, and adjust pH 7.2~7.4. If the red precipitate is still visible, repeat this step until the solution becomes clear and transparent. Typically, a small amount of residual RBCs does not affect subsequent assays (if there are few RBCs in the pellet, this step can be omitted).</li>
	</ol>
	</li>
	<li>Cell Washing: Wash the cells twice with approximately 10 mL of pre-cooled RPMI 1640 medium. Centrifuge at 500 x g for 5 min at 4 &#176;C each time and discard the supernatant.</li>
	<li>Cell resuspension and counting: Resuspend the cells in pre-cooled medium consisting of RPMI 1640 + 1 % FBS + 1% PS, count the cells using Trypan Blue staining, and assess cell viability.</li>
	<li>Cell culture: If culture is required, seed the cells at a density of 1 &#215; 106/mL in a 24-well culture plate, with 1 mL of RPMI 1640-10% FBS-1% PS medium per well. After 2 h of incubation, replace the medium and wash the wells 1-2 times with RPMI 1640 medium to remove non-adherent cells. 
	NOTE: The adherent cells will form a monolayer of macrophages. If RNA extraction is needed, add 1 mL of Trizol to the cell pellet, pipette multiple times to disrupt the cells, and transfer the mixture to a 1.5 mL microcentrifuge tube for immediate RNA extraction or store at -80 &#176;C. For FACS analysis, adjust the cell count to 1 &#215; 106 per tube, add 20 &#181;L of FACS buffer, and proceed with FACS staining. FACS buffer: DPBS + 2% FBS, filter sterilize through a 0.22 &#181;m filter, stored at 4 &#176;C).</li>
</ol>
2. Collection of rat or human RBCs
<ol>
	<li>Preparation: Pre-rinse 15 mL centrifuge tubes and 10 mL syringes with heparin.</li>
	<li>Collection of rat blood from the tail vein: After anesthetizing the rat, carefully cut a small section of the rat&#39;s tail with scissors and collect blood from the tail vein. At the end of the experiment, euthanize the rat with CO2.</li>
	<li>Collection of human blood: Collect human blood using the venipuncture technique.</li>
	<li>Mixing of blood samples: Mix 1 mL of PBS with 1 mL of human venous blood or rat blood.</li>
	<li>Ficoll density gradient separation: Add 1.5 mL of Ficoll solution with a density of 1.077 to a 15 mL centrifuge tube for human peripheral blood; add 1.5 mL of Ficoll solution with a density of 1.084 to a 15 mL centrifuge tube for rat blood.</li>
	<li>Layering of blood samples: Slowly layer the diluted blood samples along the wall of the centrifuge tube onto the corresponding density gradient solution.</li>
	<li>Centrifugation: Centrifuge at 400 x g for 30 min at a temperature of 18-20 &#176;C.</li>
	<li>Removal of plasma and lymphocyte layers: Remove the upper plasma layer, lymphocyte layer, and Ficoll solution layer, leaving only the bottom red blood cell layer.</li>
	<li>Red blood cell washing: Wash the RBCs once with 10 mL of PBS.</li>
	<li>Centrifugation and removal of supernatant: Centrifuge at 400 x g for 5 min and discard the supernatant.</li>
	<li>Cell resuspension and counting: Add 1 mL of RPMI 1640 medium to a 1.5 mL microcentrifuge tube, mix with an appropriate amount of red blood cell pellet, and perform cell counting.</li>
</ol>
3. In vitro phagocytosis assay
<ol>
	<li>Macrophage isolation and culture: Isolate macrophages and seed them at a density of 1 &#215; 106 cells/well in a 12-well culture plate. Incubate at 37 &#176;C with 5% CO2 for 2 h to allow cells to adhere.</li>
	<li>Removal of non-adherent cells: After 2 h of incubation, carefully remove non-adherent cells and gently wash with RPMI 1640 medium.</li>
	<li>Continued culture: Continue to culture the adherent macrophages for up to 2 h to ensure complete adhesion to the bottom of the culture plate.</li>
	<li>Starvation treatment: After 2 h, replace the medium with serum-free RPMI 1640 and incubate under starvation conditions for 2 h to enhance macrophage phagocytic activity.</li>
	<li>Addition of human cells: Add human cells pre-stained with DeepRed to the culture plate and incubate at 37 &#176;C with 5% CO2 for 2 h to allow macrophages to phagocytose the human cells.</li>
	<li>Conclusion and analysis: After the phagocytosis assay, observe the phagocytic activity under a microscope or quantify using flow cytometry (FACS).</li>
</ol>
4. Flow cytometry
<ol>
	<li>Cell preparation: Digest the cells with 0.05% Trypsin-EDTA and stop the digestion using RPMI 1640 medium containing 10% FBS. Centrifuge the cells, discard the supernatant, resuspend the cells in FACS buffer, and count them. Prepare 1 &#215; 106 cells per tube in two tubes. Additionally, prepare one tube of human RBCs and one tube of rat RBCs.</li>
	<li>Single-stain tube preparation: Add mouse-anti-rat CD163 antibody to one sample tube, mouse anti-human CD235a antibody to the human red blood cell tube, and DeepRed to the rat red blood cell tube.
	<ol>
		<li>Incubate these tubes on ice, protected from light, for 30 min, with mixing every 10 min. Add 1 mL of FACS buffer to wash off any unbound antibodies, centrifuge, and discard the supernatant. Resuspend the cells in 500 &#181;L of FACS buffer.</li>
	</ol>
	</li>
	<li>Sample preparation: Add mouse-anti-rat CD163, mouse anti-human CD235a antibodies, and DeepRed to the sample tubes. Incubate on ice, protected from light, for 30 min, with mixing every 10 min. Add 1 mL of FACS buffer to each sample tube to wash off any unbound antibodies, centrifuge, and remove the supernatant. Resuspend the cells in 500 &#181;L of FACS buffer.</li>
	<li>Analysis: Exclude cell adhesion by gating on FSC-H versus FSC-A. Use the single-stain tubes to adjust the appropriate voltage and compensation settings before analyzing the samples (Figure 1). Analyze the data using FlowJo v10.8.1 software. 
	NOTE: CD163-FITC and PI single-stained controls were used to set compensation.</li>
</ol>

<ol><li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NOD%2FSCID%2FGAMMAC%28null%29+mouse%3A+An+excellent+recipient+mouse+model+for+engraftment+of+human+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">NOD/SCID/GAMMAC(null) mouse: An excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li>
<li>Szpirer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+models+of+human+diseases+and+related+phenotypes%3A+A+novel+inventory+of+causative+genes%5BTitle%5D)+AND+%22Mamm+Genome%22%5BJournal%5D)">Rat models of human diseases and related phenotypes: A novel inventory of causative genes.</a> Mamm Genome. 33 (1), 88-90 (2022).</li>
<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Survival-assured+liver+injury+preconditioning+%28SALIC%29+enables+robust+expansion+of+human+hepatocytes+in+Fah+Rag2%28-%2F-%29+IL2rg%28-%2F-%29+rats%5BTitle%5D)+AND+%22Adv+Sci+%28Weinh%29%22%5BJournal%5D)">Survival-assured liver injury preconditioning (SALIC) enables robust expansion of human hepatocytes in Fah Rag2(-/-) IL2rg(-/-) rats.</a> Adv Sci (Weinh). 8 (19), e2101188(2021).</li>
<li>Rongvaux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corrigendum%3A+Development+and+function+of+human+innate+immune+cells+in+a+humanized+mouse+model%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Corrigendum: Development and function of human innate immune cells in a humanized mouse model.</a> Nat Biotechnol. 35 (12), 1211(2017).</li>
<li>Furuya, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+evidence+of+a+macrophage+barrier+in+the+xenotransplantation+of+human+hematopoietic+stem+cells+to+severely+immunodeficient+rats%5BTitle%5D)+AND+%22Xenotransplantation%22%5BJournal%5D)">The evidence of a macrophage barrier in the xenotransplantation of human hematopoietic stem cells to severely immunodeficient rats.</a> Xenotransplantation. 28 (4), e12702(2021).</li>
<li>Barclay, A. N., Van den Berg, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interaction+between+signal+regulatory+protein+alpha+%28SIRP%CE%B1%29+and+CD47%3A+Structure%2C+function%2C+and+therapeutic+target%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">The interaction between signal regulatory protein alpha (SIRPα) and CD47: Structure, function, and therapeutic target.</a> Annu Rev Immunol. 32, 25-50 (2014).</li>
<li>Ide, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+for+CD47-SIRPalpha+signaling+in+xenograft+rejection+by+macrophages%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Role for CD47-SIRPalpha signaling in xenograft rejection by macrophages.</a> Proc Natl Acad Sci U S A. 104 (12), 5062-5066 (2007).</li>
<li>Matlung, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+CD47-SIRP%CE%B1+signaling+axis+as+an+innate+immune+checkpoint+in+cancer%5BTitle%5D)+AND+%22Immunol+Rev%22%5BJournal%5D)">The CD47-SIRPα signaling axis as an innate immune checkpoint in cancer.</a> Immunol Rev. 276 (1), 145-164 (2017).</li>
<li>Oldenborg, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+CD47+as+a+marker+of+self+on+red+blood+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Role of CD47 as a marker of self on red blood cells.</a> Science. 288 (5473), 2051-2054 (2000).</li>
<li>Gu, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+quantitative+method+for+measuring+innate+phagocytosis+by+human+monocytes+using+real-time+flow+cytometry%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">A quantitative method for measuring innate phagocytosis by human monocytes using real-time flow cytometry.</a> Cytometry A. 85 (4), 313-321 (2014).</li>
<li>Joffe, A. M., Bakalar, M. H., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+phagocytosis+assay+with+reconstituted+target+particles%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Macrophage phagocytosis assay with reconstituted target particles.</a> Nat Protoc. 15 (7), 2230-2246 (2020).</li>
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JYG is a scientific advisor to Prometheus RegMed Tech Ltd. The left authors declare no potential conflict of interest.

This study established a robust and reproducible in vitro assay to evaluate macrophage-mediated phagocytosis of both xenogeneic and allogeneic RBCs. In this study, human RBCs were labeled with CD235a-BV421, while rat RBCs were stained with DeepRed dye. When macrophages phagocytose the labeled RBCs, the conjugated antibodies (CD235a-BV421) or dye (DeepRed) are internalized together. As a result, the macrophages themselves exhibit corresponding fluorescence. Under these conditions, phagocytic macrophages can be re...

The use of xenogeneic cell transplantation, particularly the engraftment of human cells into animal models, is an essential strategy in biomedical research1. It allows for the evaluation of cell survival, engraftment, and immune responses in a controlled in vivo environment. While mouse models have historically dominated this field due to their well-characterized genetics and ease of manipulation, their small size presents a significant limitation in studies requiring large blood volumes or high cell yields. In contrast, rats offer several advantages, including greater body mass, improved physiological relevance to human systems, and a higher yield of biological samples such as blood or peritoneal macrophages2. Consequently, rat models are increasingly used in studies involving hematopoietic and immune system research3.
One of the major challenges in xenogeneic transplantation is immune rejection. Even in immunodeficient models where T cells, B cells, or NK cells are genetically ablated, residual innate immune cells, particularly macrophages, remain functionally active4. Macrophages serve as first-line defenders of the host immune system, recognizing and eliminating foreign cells through phagocytosis5. This process is partly regulated by the interaction between the signal regulatory protein alpha (SIRP&#945;) on macrophages and CD47, a "don't eat me" signal expressed on most cell types. When CD47 from human cells is not recognized by rodent SIRP&#945;, this mismatch can result in enhanced phagocytosis. Even in these permissive immunodeficient animals, this phenomenon also exists6,7.
Quantifying macrophage-mediated phagocytosis in this context is critical for evaluating the likelihood of cell survival and for optimizing transplantation strategies8. Although various in vivo and ex vivo methods have been developed to assess immune rejection, standardized in vitro assays for macrophage phagocytosis remain limited. Traditional microscopy-based methods are often labor-intensive and lack quantitative rigor. Flow cytometry, by contrast, offers a single-cell-based assay and quantitative platform capable of accurately distinguishing cell types and measuring phagocytic uptake through surface marker detection.
In this article, we developed a standardized in vitro assay to evaluate the differential phagocytosis of human and rat RBCs by rat peritoneal macrophages. RBCs serve as an ideal model for phagocytosis assays due to their simple structure and expression of CD47, which acts as a "self" signal to inhibit macrophage-mediated clearance9. In this protocol, rat peritoneal macrophages are harvested via peritoneal lavage, cultured in vitro, and exposed to either human RBCs or allogeneic rat RBCs labeled with DeepRed. After co-incubation, flow cytometry is used to assess the internalization of RBCs by macrophages. Macrophages are identified by CD163 expression, human RBCs by CD235a, and rat RBCs by DeepRed fluorescence. Dead cells are excluded via PI staining, ensuring that only viable macrophages are analyzed.
This approach enables accurate, quantitative assessment of allogeneic and xenogeneic RBCs' phagocytic activity in vitro, providing insight into the extent of macrophage-mediated immune clearance. In our experiments, rat macrophages demonstrated significantly higher phagocytosis of human RBCs (5.08%) compared to rat RBCs (1.59%), yielding a relative phagocytosis index of 3.21. These results underscore the importance of innate immune mechanisms in xenogeneic settings and highlight the value of this assay in preclinical model validation.
In conclusion, the protocol presented here offers a reproducible and efficient method for measuring macrophage phagocytosis in vitro. It is particularly useful for researchers working in the fields of xenotransplantation, hematopoietic stem cell research, and immunological compatibility testing. By providing a standardized framework for phagocytosis quantification, this assay can contribute to improved experimental reproducibility and enhanced understanding of innate immune responses in cross-species transplantation models.

<table><tbody><tr><td>0.05% Trypsin-EDTA</td><td>ThermoFisher</td><td>25300062</td><td></td></tr><tr><td>Deep Red Cell Tracker</td><td>ThermoFisher</td><td>&amp;nbsp;C34565</td><td>The powder (15 &amp;mu;g) was dissolved in DMSO (20 &amp;mu;L)</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>ThermoFisher</td><td>C10010500BT</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>Cytiva</td><td>17144002</td><td></td></tr><tr><td>Ficoll-Paque PREMIUM</td><td>Cytiva</td><td>17544602</td><td></td></tr><tr><td>Mouse Anti-Human CD235a BV421 Antibody</td><td>BD Biosciences</td><td>562938</td><td></td></tr><tr><td>Mouse Anti-Rat CD163 FITC Antibody</td><td>BIO-RAD</td><td>MCA342F</td><td></td></tr><tr><td>Protein K</td><td>Sigma</td><td>P2308</td><td></td></tr><tr><td>RPMI 1640</td><td>ThermoFisher</td><td>11875093</td><td></td></tr></tbody></table>

a quantitative assessment of the phagocytosis of allogeneic and xenogeneic erythrocytes by rat macrophages in vitro

A Quantitative Assessment of the Phagocytosis of Allogeneic and Xenogeneic Erythrocytes by Rat Macrophages In Vitro

Xenogeneic cell transplantation often faces significant immune rejection, even in immunodeficient animal models. Among residual immune components, macrophages can actively phagocytose transplanted human cells, posing a challenge to long-term engraftment. To address this, we developed a standardized in vitro assay to quantify macrophage-mediated phagocytosis of human versus rat red blood cells (RBCs). Rat peritoneal macrophages were isolated, cultured, and exposed to either human or rat RBCs. Human RBCs were identified using CD235a, while rat RBCs were pre-labeled with DeepRed. Flow cytometry analysis was conducted using CD163 to identify macrophages and propidium iodine (PI) exclusion to gate living cells. The results showed a substantial difference in phagocytic activity: 5.08% of macrophages engulfed human RBCs, while only 1.59% phagocytosed rat RBCs, yielding a relative phagocytosis index of 3.21. This protocol allows for quantitative assessment of immune compatibility and offers a reproducible method to evaluate innate immune responses in xenotransplantation. It holds potential for refining donor-recipient selection and guiding immunosuppressive strategies in translational research.

Through flow cytometry analysis, we can infer the phagocytosis rate of rat macrophages towards human RBCs and rat RBCs. Dead cells are first excluded using PI. CD163-positive macrophages that express CD235a-positive indicate the phagocytosis of human RBCs. The proportion of macrophages that have phagocytosed hRBCs is defined as the phagocytosis rate of rat macrophages towards human RBCs. The proportion of phagocytosis of rat RBCs was similarly calculated. CD235a and Deep Red double-positive cells represent macrophages th...

This work describes a standardized in vitro protocol to quantify and compare the phagocytosis rates of xenogeneic (human) and allogeneic (rat) red blood cells by isolated rat macrophages.

Xenogeneic cell transplantation often faces significant immune rejection, even in immunodeficient animal models. Among residual immune components, ...

Our research aims to develop a novel strategy to quantitatively assess the in vitro phagocytosis rate of xenogeneic cells by macrophages. We designed a co-incubation system in which microphage simultaneously phagocytose both allogeneic and xenogeneic genic cells. By calculating a relative phagocytosis index, we effectively eliminated the impact of the individual variability.We have established a method for assessing the immune rejection response of macrophages toward xenogeneic cells, which offer significant advantage including broad applicability, operational simplicity and quantifiable results. Macrophages phagocytose xenogeneic cells at over three times the rate of allogeneic cells. This opens up new directions for developing strategies to xenophagocytosis, thereby improving the stability of xenotransplantation.Our laboratory will focus on developing strategies to reduce macrophage-mediated phagocytosis of xenogeneic cells and overcome immune rejection after transplantation with ultimate goal of constructing stable humanized animal models. To begin, disinfect the abdomen of the euthanized rat with ethanol and make a small incision in the abdominal wall of the rat. Using a syringe, inject 50 milliliters of ice-cold, sterile PBS into the abdominal cavity along the midline.Gently shake the rat and massage the abdominal wall with fingers to thoroughly mix the fluid in the abdominal cavity for two to three minutes. Then tilt the rat's body slightly to pool the abdominal fluid and aspirate it with a syringe. Centrifuge the collected fluid at 500 g for five minutes at four degrees Celsius.Afterward, discard the supernatant, then add three to five times the volume of the cell pellet of red blood cell lysis buffer, and gently mix for one to two minutes. After centrifuging the tube, remove the red supernatant. To prepare the red blood cell lysis buffer, mix the components shown here and adjust the volume to 1000 milliliters with distilled water.Using a 0.2 micrometer filter, filter sterilize the solution, then wash the cells twice using approximately 10 milliliters of pre-cooled RPMI-1640 medium. Centrifuge each time at 500 g for five minutes at four degrees Celsius. After discarding the supernatant, re-suspend the cells in pre-cooled medium containing RPMI-1640 with 1%FBS and 1%penicillin-streptomycin.Use trypan blue staining to count the cells and assess cell viability. If culturing is required, seed the cells in a 24-well culture plate using one milliliter of RPMI-1640 medium containing 10%FBS and 1%penicillin-streptomycin per well. After incubating for two hours, replace the medium and wash the wells one to two times with RPMI-1640 medium to remove non-adherent cells.Pre-rinse 15 milliliter centrifuge tubes and 10 milliliter syringes with heparin to prevent coagulation. After anesthetizing and euthanizing the rat, use scissors to carefully cut a small section of the rat's tail. Collect blood as it drips from the tail vein using a heparinized syringe or tube.Collect human blood using the venipuncture technique. Mix one milliliter of PBS with one milliliter of human or rat blood in a centrifuge tube. For human peripheral blood, add 1.5 milliliters of Ficoll solution with a density of 1.077 to a 15-milliliter centrifuge tube.Slowly layer the diluted blood samples along the wall of the centrifuge tubes containing the Ficoll solution to avoid mixing. Centrifuge the layered samples at 400 g for 30 minutes at a temperature between 18 and 20 degrees Celsius. Afterward, carefully remove the upper plasma layer, the lymphocyte layer, and the Ficoll solution, leaving behind only the red blood cell layer at the bottom.Wash the red blood cells once with 10 milliliters of PBS. Then centrifuge the red blood cells at 400 g for five minutes and discard the supernatant. Add one milliliter of RPMI-1640 medium to a 1.5 milliliter microcentrifuge tube.Mix with an appropriate amount of red blood cell pellet before performing cell counting. Isolate macrophages and seed them at a density of 1 million cells per well in a 12-well culture plate. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for two hours to allow cell adhesion.After two hours, carefully remove non-adherent cells and gently wash each well with RPMI-1640 medium. Replace the medium with serum-free RPMI-1640 and incubate the cells for two hours under starvation conditions to enhance phagocytic activity. Then add human cells pre-stained with Deep Red dye into the culture plate and incubate as shown earlier to allow phagocytosis by macrophages.Digest the cells using 0.05%trypsin-EDTA and stop the digestion by adding RPMI-1640 medium containing 10%FBS. Then centrifuge the cells. Discard the supernatant and re-suspend the pellet in FACS buffer.Next, prepare 1 million cells per tube in two separate tubes and additionally prepare one tube each of human and rat red blood cells. Then add mouse anti-rat CD163 antibody to one of the sample tubes. Add mouse anti-human CD235a antibody to the human red blood cell tube and add Deep Red to the rat red blood cell tube.Incubate all labeled tubes on ice, protected from light for 30 minutes with mixing every 10 minutes. Add one milliliter of FACS buffer to wash away unbound antibodies. After centrifuging the tubes, re-suspend the cell pellets in 500 microliters of FACS buffer.Exclude cell aggregates by gating on forward scatter height versus forward scatter area. Use the single-stain tubes to adjust voltage and compensation settings on the flow cytometer. Flow cytometry analysis identified CD163 positive rat macrophages with a mean frequency of 4.78%after exclusion of dead cells.The phagocytosis rate of rat macrophages toward human red blood cells was 5.08%which was higher than the rate toward rat red blood cells at 1.59%

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Research

JoVE Journal

Immunology and Infection

A novel method to obtain macrophages from primary culture of rat liver cells is described.  This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Source: Nagre, N., et al. Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice. J. Vis. Exp. (2019).This video demonstrates the assessment of complement receptor-mediated phagocytosis using sheep red blood cells opsonized with IgM and complement, followed by their uptake by mouse macrophages. The extent of phagocytosis is quantified by measuring a blue chromogenic product formed through hemoglobin-catalyzed oxidation of diaminofluorene.

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This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

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An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

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Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

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Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

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Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Learn a standardized in vitro method to quantify phagocytosis of xenogeneic and allogeneic erythrocytes by rat macrophages. This protocol establishes a relative phagocytosis index for immunology research.

A Quantitative Assessment of the Phagocytosis of Allogeneic and Xenogeneic Erythrocytes by Rat Macrophages In Vitro

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