BSRI Presentation
30
Optimizing Apoptosis Assay Rachael Jackman, PhD Sabrina Medina August 26 th , 2014
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Transcript of BSRI Presentation
Optimizing !Apoptosis Assay!
Rachael Jackman, PhD!Sabrina Medina!
August 26th, 2014!!
Background!
Objective!
Methods!
Results!
Questions!
Overview!
Background | Objective | Methods | Results !
Allogeneic transfusions!
Recipient!
Untreated !Blood!
Donor! Recipient!
Background | Objective | Methods | Results !
Allogeneic transfusions!Mirasol – UV-based pathogen reduction technology!
Background | Objective | Methods | Results !
Allogeneic transfusions!
Mirasol!Treated!Blood!
Untreated !Blood!
Mirasol – UV-based pathogen reduction technology!
Donor! Recipient!
Background | Objective | Methods | Results !
Cell Death!Apoptosis! Necrosis !
• Stimulated by physiological factors!
• Reduced (shrinks)!
• Stimulated by pathological factors!
• Enlarged (swells)!
• Greater membrane integry!
• Ruptures!
• Cell SUICIDE! • Cell HOMOCIDE!
Objective!
To develop and optimize an apoptosis assay!to help decipher mechanisms that regulate
alloresponse to pathogen-reduced platelets in order to improve outcomes of allogeneic transfusions in
patients. !
Background | Objective | Methods | Results !
Objective!
2. Establish controls for cell death!• Used to compare to Mirasol treated cells to induce similar cell
death occuring in white blood cells !• By testing known apoptosis/necrosis inducers!
1. Establish an assay for apoptosis!• Evaluate how Mirasol treated cells are dying!• By looking at different dyes to evaluate cell characteristics !
Background | Objective | Methods | Results !
Background | Objective | Methods | Results !
General Protocol!1. Cell Harvest • B6 Mice !• Splenocytes / PBMC!
2. Treatments • Apoptosis: Dexamethasone, 5% Ethanol!• Necrosis: 10% Ethanol, Heat, Freeze/Thaw!• Apoptosis/Necrosis?: Mirasol!
3. Staining • Propidium Iodide, SYTOX, Annexin V, CellEvent, Vybrant DyeCycle!
3. FACS Analysis
Stains!
Background | Objective | Methods | Results !
PI!
PI!
Annexin V!APC!
Annexin V!APC!
Membrane Integrity & Phosphatidylserine Exposure!
Phosphotidylserine !
Normal Cell!
Annexin V!
Background | Objective | Methods | Results !
Membrane Integrity & Phosphatidylserine Exposure!
PI!
PI!
Annexin V!APC!
APC!Early Apoptosis!
Annexin V!
Background | Objective | Methods | Results !
Membrane Integrity & Phosphatidylserine Exposure!
PI!
PI!
Annexin V!APC! APC!
Early Apoptosis!
Annexin V!
Background | Objective | Methods | Results !
Membrane Integrity & Phosphatidylserine Exposure!
PI! PI!
Annexin V!APC!
APC!Late Apoptosis / Necrosis!
Background | Objective | Methods | Results !
Membrane Integrity & Phosphatidylserine Exposure!
Caspase Activity!Peptide! Dye
Peptide! Dye!
Peptide! Dye!
Caspase 3/7!
CellEvent!
Background | Objective | Methods | Results !
Caspase Activity!Peptide! Dye
Peptide! Dye!
Peptide!
Dye!
Caspase 3/7!
CellEvent!
Background | Objective | Methods | Results !
Chromatin Condensation!Vybrant DyeCycle!
Background | Objective | Methods | Results !
Chromatin Condensation!Vybrant DyeCycle!
Background | Objective | Methods | Results !
Process!
Apoptotic treatment
control!
Applying to pathogen-
reduced cells!
Background | Objective | Methods | Results !
Background | Objective | Methods | Results !
5% Ethanol! Dexamethasone (steroid)!
10% Ethanol!
Establishing Apoptotic Treatment Control: 5% Ethanol!
Background | Objective | Methods | Results !
Pathogen Reduced Cells – Membrane Integrity & PS Exposure!
PI low!
PI negative!
Background | Objective | Methods | Results !
Pathogen Reduced Cells – Chromatin Condensation & PS Exposure!
Background | Objective | Methods | Results !
Pathogen Reduced Cells – Chromatin Condensation & PS Exposure!
? PI low!
PI negative!
Background | Objective | Methods | Results !
Pathogen Reduced Cells – Caspase Activity in Live Gate!
Mirasol!Freeze / Thaw!5% Ethanol!Untreated!
General Gating Strategy!
Background | Objective | Methods | Results !
Pathogen Reduced Cells – Caspase Activity in Apoptotic Gate!
Mirasol!Freeze / Thaw!5% Ethanol!Untreated!
General Gating Strategy!
Background | Objective | Methods | Results !
Pathogen Reduced Cells – Caspase Activity in Dead Gate!
Mirasol!Freeze / Thaw!5% Ethanol!Untreated!
General Gating Strategy!
Conclusion!Mirasol treated cells are not classically apoptotic !
• PS Exposure? !!• Cell permeable? !
• Chromatin Condensation?!
!• Caspase 3/7 Activation? !
Future research!
• Different time points for data collection after treatment! !
• Stain for apoptosis initiator caspases!
?
Acknowledgements!Rachael Jackman!Dale Hirschkorn !
Phillip Norris !Heather Inglis !
Marcus Muench!Heather Inglis !Ashley Beyer!
Fredda Assenzio!Jessica Bailey!
!!
Questions!
