To determine the phagocytic capacity of macrophages, pHrodo Red S. aureus bioparticles conjugate (Invitrogen, Carlsbad, CA) was added to the culture media according to the manufacturer's recommendations with ~2 × 106 cells per 2 mg vial of pHrodo-labeled bioparticles. Cells were incubated with the pHrodo labeled bioparticles for 2 hrs and then collected for FACS analysis and data analysis by Flowjo. This phagocytosis assay is based on the fact that there is a minimal fluorescence signal when the pHrodo Red S. aureus bioparticle conjugate is adherent to the outer surface of the phagocyte. Once the S. aureus is internalized and incorporated into the acidic environment of the phagosome, the bioparticle conjugates emit a strong red fluorescence. Internalization was verified by live cell confocal imaging (Olympus FluoView FV1000, Center Valley, PA). To assess changes in viability due to ethanol, MH-S cells were stained with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and Propidium Iodide (PI) (Invitrogen, Carlsbad, CA) before analysis by flow cytometry.
Dead cell apoptosis kit with annexin 5 alexa fluor 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor® 488 is a laboratory tool used to detect and quantify apoptosis, or programmed cell death, in cell samples. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a molecule that is exposed on the cell surface during apoptosis. The Annexin V is conjugated with the Alexa Fluor® 488 fluorescent dye, allowing for the detection of apoptotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (8 mentions)
Brdu cell proliferation assay, by Cell Signaling Technology (2 mentions)
Glomax discover, by Promega (2 mentions)
Cell proliferation kit 1 mtt assay, by Merck Group (2 mentions)
Phrodo red s aureus bioparticles conjugate, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Caspase glo 3 7 assay system, by Promega (1 mentions)
Caspase glo 8 assay system, by Promega (1 mentions)
Caspase glo 9 assay system, by Promega (1 mentions)
Fas associated protein with death domain fadd, by Cell Signaling Technology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Kaempferol, by Merck Group (1 mentions)
Tgf β1, by Sino Biological (1 mentions)
Lipofectamine imax, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Itaq universal sybr green one step kit, by Bio-Rad (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Facscanto 2 analyzer, by BD (1 mentions)
9 protocols using dead cell apoptosis kit with annexin 5 alexa fluor 488
1
Phagocytic Capacity of Macrophages
2
Evaluating Breast Cancer Cell Viability
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For determining cell viability, 50,000 MDA-MB-231 cell variants were plated into a 6-well plate. At 72 h, cells were trypsinized, harvested, stained with trypan blue, and counted using a hemocytometer. Cells were deemed viable upon trypan blue exclusion. The Cell Proliferation Kit I MTT Assay (Millipore Sigma, St. Louis, MO, USA) was also used to evaluate tumor cell viability over time. 2000 MDA-MB-231, MDA-MB-468, or MVT1 variants were plated and measurements taken at 72 h using a Promega Glomax Discover microplate reader. For DMSO, BQU57, and Taxol treatments, the media contained drugs at the indicated concentration and measurements were taken after 72 h.
The BrdU Cell Proliferation Assay (Cell Signaling, Danvers, MA, USA) was used to evaluate tumor cell proliferation. 10,000 MDA-MB-231 or 5000 MVT1 cell variants were plated and the assay was performed following manufacturer’s instructions. At 72 h, absorbance was read at 450 nm using a Promega Glomax Discover microplate reader.
The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 (Invitrogen, Carlsbad, CA, USA) was used to evaluate apoptotic populations. MDA-MB-231 or MVT1 cell variants were plated and allowed to reach ~90% confluency. Floating and harvested cells were collected and treated according to manufacturer’s instructions. Cells were acquired using a LSR II.
The BrdU Cell Proliferation Assay (Cell Signaling, Danvers, MA, USA) was used to evaluate tumor cell proliferation. 10,000 MDA-MB-231 or 5000 MVT1 cell variants were plated and the assay was performed following manufacturer’s instructions. At 72 h, absorbance was read at 450 nm using a Promega Glomax Discover microplate reader.
The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 (Invitrogen, Carlsbad, CA, USA) was used to evaluate apoptotic populations. MDA-MB-231 or MVT1 cell variants were plated and allowed to reach ~90% confluency. Floating and harvested cells were collected and treated according to manufacturer’s instructions. Cells were acquired using a LSR II.
3
Comprehensive Viability and Proliferation Assays
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For determining cell viability, 50,000 MDA-MB-231 cell variants were plated into a 6-well plate. At 72 hours, cells were trypsinized, harvested, stained with trypan blue, and counted using a hemocytometer. Cells were deemed viable upon trypan blue exclusion. The Cell Proliferation Kit I MTT Assay (Millipore Sigma, St. Louis, MO, USA) was also used to evaluate tumor cell viability over time. 2000 MDA-MB-231, MDA-MB-468, or MVT1 variants were plated and measurements taken at 72 hours using a Promega Glomax Discover microplate reader. For DMSO, BQU57, and Taxol treatments, the media contained drugs at the indicated concentration and measurements were taken after 72 hours.
The BrdU Cell Proliferation Assay (Cell Signaling, Danvers, MA, USA) was used to evaluate tumor cell proliferation. 10,000 MDA-MB-231 or 5000 MVT1 cell variants were plated and the assay was performed following manufacturer's instructions. At 72 hours, absorbance was read at 450 nm using a Promega Glomax Discover microplate reader.
The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 (Invitrogen, Carlsbad, CA, USA) was used to evaluate apoptotic populations. MDA-MB-231 or MVT1 cell variants were plated and allowed to reach ~ 90% confluency. Floating and harvested cells were collected and treated according to manufacturer's instructions. Cells were acquired using a LSR II.
The BrdU Cell Proliferation Assay (Cell Signaling, Danvers, MA, USA) was used to evaluate tumor cell proliferation. 10,000 MDA-MB-231 or 5000 MVT1 cell variants were plated and the assay was performed following manufacturer's instructions. At 72 hours, absorbance was read at 450 nm using a Promega Glomax Discover microplate reader.
The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 (Invitrogen, Carlsbad, CA, USA) was used to evaluate apoptotic populations. MDA-MB-231 or MVT1 cell variants were plated and allowed to reach ~ 90% confluency. Floating and harvested cells were collected and treated according to manufacturer's instructions. Cells were acquired using a LSR II.
4
Kaempferol Induces Apoptosis in Cancer Cells
5
Quantitative Gene Expression Analysis Protocol
6
Apoptosis Detection by Annexin V
7
Measuring Apoptosis with Annexin V-Alexa Fluor 488
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Steady‐state apoptosis assays were performed with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 and propidium iodide (PI) obtained from Thermo Fisher Scientific (#V13241), and assays were performed according to manufacturer's protocol. Briefly, approx. 106 cells were seeded in a 60‐mm cell culture plate and incubated at 37°C in an incubator as normal. Subsequently, the cells were harvested with trypsin–EDTA after overnight growth (between 16 and 18 h) and stained with annexin V conjugated with Alexa 488. For detection of dead/necrotic cells, PI counterstaining was also performed. Negative staining controls and single dye staining controls were also made for each cell type for offline gating analyses with FlowJo®.
All samples were processed in a BD FACSCanto II analyzer.
All samples were processed in a BD FACSCanto II analyzer.
8
Fisetin's Cytotoxic Effects via Apoptosis Pathway
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Fisetin (purity ≥ 98%), thiazolyl blue tetrazolium bromide (MTT) and mouse monoclonal β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sorafenib Tosylat N Mikron (BAY 43-9006) was provided by Bayer HealthCare (Bayer Pharma AG, Berlin, Germany). Dead cell apoptosis kit with annexin V alexa fluor® 488 and propidium iodide (PI) were obtained from Life Technologies (Grand Island, NY, USA). Rabbit monoclonal or polyclonal antibodies for PARP, cleaved caspase-3, Bcl2, Bax, Bak, Mcl-1, PI3Kp110α, PI3Kp85, pAKT Ser473, pmTOR Ser2448, pmTOR Ser2481, pMEK1/2 and pERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit monoclonal cyclin D1 antibody was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Rabbit polyclonal Ki67, goat polyclonal PNCA, goat polyclonal PECAM-1/CD31 and mouse monoclonal VEGF antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase conjugates goat anti-rabbit, rabbit anti-goat and rabbit anti-mouse antibodies were purchased from Millipore Corporation (Billerica, MA, USA). Alexa Fluor 488 rabbit anti-goat antibodies were procured from Life Technologies (Grand Island, NY, USA).
9
Analyzing Cell Viability and Apoptosis in OSKM Reprogramming
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