Cell cytotoxicity / Proliferation assay cell type - K562

Cell cytotoxicity assays measure the ability of certain compounds or chemical mediators to reduce the viability of the cells. The term cell cytotoxicity assay can sometimes be used interchangeably with cell proliferation assay. Healthy living cells can be identified by the use of formazan dyes, protease biomarkers or by measuring ATP content. The formazan dyes are chromogenic products formed by the reduction of tetrazolium salts by dehydrogenases, such as lactate dehydrogenase (LDH) and reductases that are released during cell death. Common tetrazolium salts include INT, MTT, MTS and XTT. Cell cytotoxicity can also be measured by using the SRB and WST-1 assays. These assays can usually be used in a high-throughput fashion and can be quantitated by measuring absorbance, colorimetry or luminescence. All these assays require similar numbers of cell plating at the initiation, a time course of treatment with the cytotoxic agent and at least triplicates for each condition at every point of analysis. Cell shrinkage, plasma membrane blebbing, cell detachment, externalization of phosphatidylserine, nuclear condensation and ultimately DNA fragmentation are well-described features of apoptosis. The assays that rely on cell membrane integrity for their function, may not be able to quantify early apoptosis. Therefore, in order to distinguish early apoptotic vs. late apoptotic or necrotic cells, additional flow cytometry techniques can be used. A combination of Annexin V and PI (propidium iodide) can be used to distinguish early (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells. Sometimes, caspase assays are used in order to differentiate the stages of apoptosis.

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Found 4 matching solutions for this experiment

LC3A/B (D3U4C) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45
µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X
transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was
blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were
then washed three times, five minutes each, with 1X TBST.
Protocol tips
Primary incubation with
antibodies was done overnight at 4°C. After primary incubation, the blots were washed
five times, five minutes each, with 1X TBST and then incubated in secondary antibody in
1% milk TBST for thirty minutes at room temperature. After secondary antibody
incubation, the blots were washed five times, five minutes each, with 1X TBST and then
once with 1X TBS for ten minutes.
SQSTM1 Antibody (D-3)

Santa Cruz Biotechnology

Upstream tips
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45
µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X
transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was
blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were
then washed three times, five minutes each, with 1X TBST.
Protocol tips
Primary incubation with
antibodies was done overnight at 4°C. After primary incubation, the blots were washed
five times, five minutes each, with 1X TBST and then incubated in secondary antibody in
1% milk TBST for thirty minutes at room temperature. After secondary antibody
incubation, the blots were washed five times, five minutes each, with 1X TBST and then
once with 1X TBS for ten minutes.
SQSTM1/p62 Antibody

Cell Signaling Technology

Upstream tips
Cells were lysed in RIPA buffer with PMSF (PMSF:RIPA=1:100) for 30 min on ice and then centrifuged for 15 min. Protein concentration of the supernatant was measured using a BCA protein assay kit.
Protocol tips
Equal amounts of protein from cell extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (30). The membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with horseradish peroxidase.
Downstream tips
Finally, the protein level was determined using a chemiluminescent approach in a dark room.
Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

Upstream tips
Cells were lysed in RIPA buffer with PMSF (PMSF:RIPA=1:100) for 30 min on ice and then centrifuged for 15 min. Protein concentration of the supernatant was measured using a BCA protein assay kit.
Protocol tips
Equal amounts of protein from cell extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (30). The membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with horseradish peroxidase.
Downstream tips
Finally, the protein level was determined using a chemiluminescent approach in a dark room.
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