ORFLO Technologies Ketchum, ID [email protected] 855-TRY-MOXI www.orflo.com Orflo Application Protocol 05/2015 Moxi Flow Apoptosis (PE-Annexin V) and Viability (PI) - Staining Protocol Page 1 Reagents: • Annexin V Binding Buffer (BioLegend, cat#422201) • Cell Staining Buffer (BioLegend, cat#420201) • PE-Annexin V conjugate (BioLegend, cat#640907) • Orflo Flow Reagent (Orflo Technologies, MXA080) • Moxi Cyte Viability Kit (Orflo Technologies, MXA055) Protocol 1. Induce apoptosis in cells by desired method (e.g. 20μM Camptothecin treated, 6+ hours, 37°C) Jurkat cells. Include a control sample of untreated cells. 2. Isolate cells to a single-cell suspension. (If necessary use a protease and/or pipette trituration to break apart the clusters) 3. Wash cells twice in Cell Staining Buffer (2.5mL volume, 300 x g, 5 minutes). 4. Re-suspend pellet to 1 x 10 6 cells/ml in Annexin V Binding Buffer (verify counts with the Moxi Z or Moxi Flow instruments). 5. For each sample, prepare two microcentrifuge tubes. Note: the tubes can be processed concurrently to reduce overall processing time. • Tube 1 (PE- Annexin V measurement): a. Add 100µl of cells b. Add 10 μl of PE-Annexin V conjugate. Notes: Required PE-Annexin V concentration may vary by PE-Annexin manufacturer. We recommend 0.3 – 0.6µg/ml final PE – Annexin V concentration. c. Gently vortex the cells and incubate for 15 minutes at room temperature (25°C), protected from light. d. Wash cells twice (1mL volume, 300 x g, 5 minutes). with Annexin V Binding Buffer (critical, using a low Ca 2+ or Ca 2+ -free buffer will disrupt Annexin binding) e. Re-suspend pellet in 400µL of Annexin V Binding Buffer f. Add 8 uL of Moxi Cyte Flow Reagent. Vortex to mix. g. Run on Moxi Flow using the “Apoptosis (Annexin V-PE)” app within 2 hours of staining, protect from light. • Tube 2 (PI/Viability measurement): a. Add 60µL of cell suspension to 240µL of Moxi Cyte Viability Kit (5x dilution) b. Gently vortex the cells and incubate for 5 minutes at room temperature (25°C), protected from light. c. Run on Moxi Flow using the “Viability Count (PI)” app within 2 hours of staining, protect from light. 6. As all membrane-permeable (dead) cells will stain positive for PE-Annexin V, the early- stage apoptosis percentage can be calculated as the PE-Annexin V positive percentage (from the first test) minus the PI positive percentage (from the second test).

Moxi Flow - PE-Annexin V (and PI) - Apoptosis - Protocol Flow - PE...Microsoft Word - Moxi Flow - PE-Annexin V (and PI) - Apoptosis - Protocol.docx Created Date 7/24/2016 4:00:55 AM

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Page 1: Moxi Flow - PE-Annexin V (and PI) - Apoptosis - Protocol Flow - PE...Microsoft Word - Moxi Flow - PE-Annexin V (and PI) - Apoptosis - Protocol.docx Created Date 7/24/2016 4:00:55 AM

ORFLO Technologies Ketchum, ID [email protected] 855-TRY-MOXI www.orflo.com

O r f l o A p p l i c a t i o n P r o t o c o l 0 5 / 2 0 1 5

MoxiFlowApoptosis(PE-AnnexinV)andViability(PI)-StainingProtocol

Page1

Reagents:

• AnnexinVBindingBuffer(BioLegend,cat#422201)• CellStainingBuffer(BioLegend,cat#420201)• PE-AnnexinVconjugate(BioLegend,cat#640907)• OrfloFlowReagent(OrfloTechnologies,MXA080)• MoxiCyteViabilityKit(OrfloTechnologies,MXA055)

Protocol1. Induceapoptosisincellsbydesiredmethod(e.g.20μMCamptothecintreated,6+hours,37°C)

Jurkatcells.Includeacontrolsampleofuntreatedcells.2. Isolatecellstoasingle-cellsuspension.(Ifnecessaryuseaproteaseand/orpipettetrituration

tobreakaparttheclusters)3. WashcellstwiceinCellStainingBuffer(2.5mLvolume,300xg,5minutes).4. Re-suspendpelletto1x106cells/mlinAnnexinVBindingBuffer(verifycountswiththeMoxi

ZorMoxiFlowinstruments).5. Foreachsample,preparetwomicrocentrifugetubes.Note:thetubescanbeprocessed

concurrentlytoreduceoverallprocessingtime.• Tube1(PE-AnnexinVmeasurement):

a. Add100µlofcellsb. Add10μlofPE-AnnexinVconjugate.Notes:RequiredPE-AnnexinVconcentration

mayvarybyPE-Annexinmanufacturer.Werecommend0.3–0.6µg/mlfinalPE–AnnexinVconcentration.

c. Gentlyvortexthecellsandincubatefor15minutesatroomtemperature(25°C),protectedfromlight.

d. Washcellstwice(1mLvolume,300xg,5minutes).withAnnexinVBindingBuffer(critical,usingalowCa2+orCa2+-freebufferwilldisruptAnnexinbinding)

e. Re-suspendpelletin400µLofAnnexinVBindingBufferf. Add8uLofMoxiCyteFlowReagent.Vortextomix.g. RunonMoxiFlowusingthe“Apoptosis(AnnexinV-PE)”appwithin2hoursof

staining,protectfromlight.• Tube2(PI/Viabilitymeasurement):

a. Add60µLofcellsuspensionto240µLofMoxiCyteViabilityKit(5xdilution)b. Gentlyvortexthecellsandincubatefor5minutesatroomtemperature(25°C),

protectedfromlight.c. RunonMoxiFlowusingthe“ViabilityCount(PI)”appwithin2hoursofstaining,

protectfromlight.6. Asallmembrane-permeable(dead)cellswillstainpositiveforPE-AnnexinV,theearly-

stageapoptosispercentagecanbecalculatedasthePE-AnnexinVpositivepercentage(fromthefirsttest)minusthePIpositivepercentage(fromthesecondtest).

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