LIVE/DEAD™ Cell-Mediated Cytotoxicity Kit, for animal cells

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Incubate cells in staining solution overnight under normal culture conditions.	Effector cells were added at effector-to-target (E:T) ratios of 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, and 80:1.

Upstream tips
Incubate cells in staining solution overnight under normal culture conditions.
Protocol tips
Effector cells were added at effector-to-target (E:T) ratios of 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, and 80:1.

Publication protocol

Assays were performed using the flow-cytometry-based nonradioactive Live/Dead Cell Mediated Cytotoxicity Kit (Invitrogen), using 5×103 targets per well labeled with 3,3′-dioctadecyloxacarbocyanine (DiOC). Target cells used included CD19-positive Raji cells, CD19-negative K562 cells, and CD19-positive K562 cells (CD19-K562). Effector cells were added at effector-to-target (E:T) ratios of 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, and 80:1. Cell mixtures were incubated in tissue-culture-treated U-bottom 96-well plates in 5% CO2 at 37°C in propidium iodide (PI)-containing R10 for times described in the Results section. Nonviable targets were double-positive cells for DiOC and PI. An LSRII cytometer was used to acquire all samples, and percent lysis of targets was based upon the following equation: Percent of specific lysis=[no. of nonviable target cells in coculture with effector cells/(no. of nonviable target cells in coculture+no. of viable target cells in coculture)] – [no. of nonviable targets cultured alone/(no. of nonviable targets cultured alone+no. of viable targets cultured alone)]×100% (Kane et al., 1996). All samples were loaded in triplicate, and all assays had controls with isolated effectors and targets. rhuGM-CSF was added at the concentration of 10 ng/ml to the assay medium in some experiments.

Apoptosis induction was evaluated by using the flow-cytometry-based FITC Annexin V Apoptosis Detection Kit I (BD Biosciences). Effector cells were incubated in R10 with target Raji cells at an E:T ratio 10:1 in tissue-culture-treated U-bottom 96-well plates in 5% CO2 at 37°C for 6 hr. Staining with the kit reagents accordingly to instructions was followed by flow cytometry acquisition within 1 hour. Target cells were identified by costaining with an APC-labeled murine monoclonal antibody to human CD19 (BD Biosciences).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Live / Dead assay mammalian cells - K562 using LIVE/DEAD™ Cell-Mediated Cytotoxicity Kit, for animal cells from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for LIVE/DEAD™ Cell-Mediated Cytotoxicity Kit, for animal cells below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Live / Dead assay mammalian cells - K562 using LIVE/DEAD™ Cell-Mediated Cytotoxicity Kit, for animal cells from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.