Establishing staining parameters for successfully discriminating target cells from effector cells using flow cytometry
Presenter Type
UNO Undergraduate Student
Major/Field of Study
Biology
Other
Biology
Advisor Information
Dr. Paul Denton
Location
MBSC Ballroom Poster # 405 - U
Presentation Type
Poster
Start Date
24-3-2023 10:30 AM
End Date
24-3-2023 11:45 AM
Abstract
Establishing staining parameters for successfully discriminating target cells from effector cells using flow cytometry
Arriana Blackmon1, Anna R. Mahr1, Maia Bennett1, and Paul W. Denton, PhD1
1 Department of Biology, University of Nebraska at Omaha, Omaha, NE
Background: The Denton Immunobiology laboratory has an overall research goal of improving human immune functions - with a particular emphasis on natural killer (NK) cells. As their name implies, NK cells kill target cells (e.g., infected or malignant cells). To study human NK cell killing activity ex vivo, our laboratory has developed a specialized technique we call the Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA). This name is given because we can use this strategy to test NK cell killing following two distinct mechanisms of target cell recognition (direct or via antibody “bridging”). Our assay requires that we be able to clearly distinguish targets from effectors (the NK cells) when we assess killing efficacy. Developing and optimizing that method is the focus of this abstract. We assess killing using a flow cytometer. Therefore, we sought a fluorescent strategy to separate targets from effectors. A cost effective and efficient method for causing cells to fluoresce is staining with CFDA-SE. When this molecule is taken into cells, the molecule is converted to CFSE a protein dye that fluoresces green when excited by blue light. We determined to apply this dye to our target cells such that we can focus our analyses on “green” cells to determine their viability within the NK-SADKA. For this to work, we needed to determine parameters (e.g., concentration) for how to treat target cells with CFDA-SE to achieve optimal fluorescence (e.g., ~ 1 log higher than background). We tested staining characteristics for two different target cells (i.e., lymphoma cells as ADCC targets and leukemia cells as direct killing targets). Our data support both cell types being labeled with 1.2 μM CFDA-SE to achieve optimum fluorescence. This finding allows us to implement our NK-SADKA in many context, including assessing the impact of interventions (e.g., immunotherapies) on the ability of human NK cells to mediate ADCC as well as direct killing of target cells.
Scheduling
10:45 a.m.-Noon, 1-2:15 p.m.
Establishing staining parameters for successfully discriminating target cells from effector cells using flow cytometry
MBSC Ballroom Poster # 405 - U
Establishing staining parameters for successfully discriminating target cells from effector cells using flow cytometry
Arriana Blackmon1, Anna R. Mahr1, Maia Bennett1, and Paul W. Denton, PhD1
1 Department of Biology, University of Nebraska at Omaha, Omaha, NE
Background: The Denton Immunobiology laboratory has an overall research goal of improving human immune functions - with a particular emphasis on natural killer (NK) cells. As their name implies, NK cells kill target cells (e.g., infected or malignant cells). To study human NK cell killing activity ex vivo, our laboratory has developed a specialized technique we call the Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA). This name is given because we can use this strategy to test NK cell killing following two distinct mechanisms of target cell recognition (direct or via antibody “bridging”). Our assay requires that we be able to clearly distinguish targets from effectors (the NK cells) when we assess killing efficacy. Developing and optimizing that method is the focus of this abstract. We assess killing using a flow cytometer. Therefore, we sought a fluorescent strategy to separate targets from effectors. A cost effective and efficient method for causing cells to fluoresce is staining with CFDA-SE. When this molecule is taken into cells, the molecule is converted to CFSE a protein dye that fluoresces green when excited by blue light. We determined to apply this dye to our target cells such that we can focus our analyses on “green” cells to determine their viability within the NK-SADKA. For this to work, we needed to determine parameters (e.g., concentration) for how to treat target cells with CFDA-SE to achieve optimal fluorescence (e.g., ~ 1 log higher than background). We tested staining characteristics for two different target cells (i.e., lymphoma cells as ADCC targets and leukemia cells as direct killing targets). Our data support both cell types being labeled with 1.2 μM CFDA-SE to achieve optimum fluorescence. This finding allows us to implement our NK-SADKA in many context, including assessing the impact of interventions (e.g., immunotherapies) on the ability of human NK cells to mediate ADCC as well as direct killing of target cells.