The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity

Gillissen, Marijn A.; Yasuda, Etsuko; Jong, Greta de; Levie, Sophie E.; Go, Daniel; Spits, Hergen; Helden, Pauline M. van; Hazenberg, Mette D.

doi:10.1016/j.jim.2016.04.002

Cited by 33 publications

(21 citation statements)

References 18 publications

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“…We determined antibody-dependent cellular cytotoxicity (ADCC) as described before (39). Briefly, PBMCs were thawed and rested overnight in IMDM with 8% FCS at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells

Cortjens

Yasuda

et al. 2017

J Virol

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Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research, and in human serum, these antibodies contribute to Ͼ90% of the neutralization response; however, detailed insight into the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of three healthy donors for specificity against RSV, CD27 ϩ memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells, 0.35% recognized RSV-A2-infected cells, of which 59% were IgAexpressing cells and 41% were IgG-expressing cells. When we generated monoclonal B cells selected for high binding to RSV-infected cells, 44.5% of IgGexpressing B cells and 56% of IgA-expressing B cells reacted to the F protein, while, unexpectedly, 41.5% of IgG-expressing B cells and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predicted cross-reactivity between RSV strain A (RSV-A) and RSV-B and matched the potency of antibodies to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of RSV-A2-infected cells. However, these processes did not seem to depend on a specific epitope. In conclusion, healthy adults harbor a diverse repertoire of RSV glycoprotein-specific antibodies with a broad range of effector functions that likely play an important role in antiviral immunity.IMPORTANCE Human RSV remains the most common cause of severe lower respiratory tract disease in premature babies, young infants, the elderly, and immunocompromised patients and plays an important role in asthma exacerbations. In developing countries, RSV lower respiratory tract disease has a high mortality. Without an effective vaccine, only passive immunization with palivizumab is approved for prophylactic treatment. However, highly potent RSV-specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition, these antibodies could guide further vaccine development. In this study, we isolated and characterized several novel antibodies directed at the RSV G protein. This information can add to our understanding and treatment of RSV disease.

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“…We determined antibody-dependent cellular cytotoxicity (ADCC) as described before (39). Briefly, PBMCs were thawed and rested overnight in IMDM with 8% FCS at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells

Cortjens

Yasuda

et al. 2017

J Virol

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“…34 Incubation of the AML cell line SH2 with AT1413 and human peripheral blood mononuclear cells or rabbit complement-induced ADCC and CDC, respectively ( Figure 4A). Endothelial cells (HAEC or HUVEC) and granulocytes, which bound AT1413, albeit to a lesser extent than AML cells, were not killed when incubated with AT1413 and peripheral blood mononuclear cells ( Figure 4B).…”

Section: At1413 Induces Adcc and Cdc Of Aml Blasts But Not Nonmalignmentioning

confidence: 99%

Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice

et al. 2017

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“…Single cell sorting is a vital part of flow core laboratories, which is often utilized by researchers for oncological, immunological, and cell line development studies . The current single cell sorting verification method commonly uses a light microscope to manually identify the existence of single cells after settling or a single colony after days of outgrowth .…”

Section: Discussionmentioning

confidence: 99%

“…Cell sorting is one of the most important methodologies required for research fields such as immunology, oncology, protein/cell engineering, neuroscience, and stem cell research . Specifically, single cell sorting is commonly used to ensure monoclonality and to produce homogenous cell populations for cell line development .…”

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confidence: 99%

A rapid single cell sorting verification method using plate‐based image cytometry

Zigon

Purseglove²,

Toxavidis

et al. 2018

Cytometry Pt A

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Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.

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The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity

Cited by 33 publications

References 18 publications

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells

Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice

A rapid single cell sorting verification method using plate‐based image cytometry

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