Apoptosis Assay

Apoptosis, or programmed cell death, is the process by which cells regulate how they die, activating specific pathways that cause the cell to shrink, condense, and eventually be cleared by phagocytosis. This is in contrast to necrotic cell death where cells die uncontrollably and fall apart, which can lead to detrimental effects such as the activation of an immune response. Therefore, apoptotic cells that die in a very orderly fashion limit disruption of nearby cells and tissue. There are many ways to measure cell death and distinguish it from apoptosis or necrosis. These assays are easily quantified using the NovoCyte flow cytometer due to automatic compensation settings and a wide dynamic range of fluorescence detection which eliminates the need for any PMT voltage adjustments.

Immunophenotyping

Immune status is associated with disease state, treatment efficiency, and response to external stimuli such as vaccines. Immunophenotyping quickly identifies candidate cell types, sub-classes and functions. Monitoring the frequency of numerous immune cell population as well as the differentiation/activation status of specific cell subsets such as monocytes, NK cells, T and B cells is essential as they may influence the immunogenicity of a vaccine and its efficiency. The NovoCyte Flow Cytometer enables simultaneous quantification of multiple leukocytes for better understanding the immune status of patients and surveillance of the immune response to infectious disease.

Specifcity	Clone	Fluorocrome	Purpose
CD3	UCHT1	PE-TR (ECD)	Lineage T cells
CD4	S3.6	PE-Alexa 700	Lineage T cells
CD8	SK1	PerCP-Cy5.5″	Lineage T cells
CD19	J3-129	PerCP-eFluor 710	B cells
CD14	MφP9	BV711	Monocytes
CD56	HCD56	BV605	NK cells and NK T-like cells
CD16	3G8	APC-Cy7	NK cells and monocytes
γδ TCR	11F2	PE-Cy7	γδ T cells
Vγ2 TCR	B6	PE	γδ T cells
CD25	M-A251	BV421	Tregs
CD127	A019D5	APC	Tregs/memory/differentiation
CD45RA	HI100	BV650	Memory/differentiation
CCR7	G043H7	BV785	Memory/differentiation
CD57	NK-1	FITC	Memory/differentiation
HLA-DR	B169414	BV570	Activation
CD38	HIT2	PE-Cy5	Activation/plasmablasts
NKG2C	134591	Alexa 700	NK receptor
Dead Cells	134591	AViD	Dead cell exclusion

Intracellular Protein Detection

Detection and analysis of intracellular proteins allow for additional characterization of cell subpopulations and cellular processes. In order to analyze proteins not located on the cell surface, fixation and permeabilization of the cell is required. However, many phospho-specific antibodies are not compatible with many common detergent-based permeabilization methods used for intracellular staining. Special attention is needed when determining the proper fix/perm method for your phospho-specific antibody. The most common method uses 1.5% paraformaldehyde for fixation followed by 100% methanol for permeabilization. While this method works for many antibodies, please note it may not work for every phospho-specific antibody. Additionally, identifying various cell populations in a heterogenous sample requires staining for phosphorylated proteins coupled with surface proteins. Special consideration must be given to the sensitivity of these epitopes to fixative, taking precaution to avoid damage to the epitope. Therefore, the sample may require staining for specific surface markers before fixation.  

Cell Cycle Analysis

Normal human somatic cells are diploids containing a constant amount of DNA. During cell cycle progression, DNA synthesis results in a doubling of total DNA content, followed by restoration of the normal DNA content after mitosis. Detailed cell cycle analysis can be performed to understand tumor cell differentiation, cell transformation and cell-compound interaction with the NovoCyte flow cytometer. Figure: After treatment with 10 migrograms/M MG132 or 500 micrograms/M 5-FI for 16 hours/ A549 cells were analyzed for cell cycle distribution with the ACEA Novocyte flow cytometer. The the Novoexpress built-in cell cycle analysis module, the plot shows cells ni G0/G1 phase (green), S phase (yellow) and G2/M phase (blue). Compared to normal untreated cells, MG132 treated cells were arrested at G2/M phase, while 5-FU treated cells were arrested at G0/G1 phase.  

Cell Proliferation

Cell proliferation is an essential function and highly structured event that when unregulated, can cause disease. We can measure proliferation through absolute cell counts or with a dye, such as CFSE. When cells labeled with CFSE divide, the dye is partitioned equally between daughter cells and we can measure the loss of CFSE fluorescence over time as the dye is continuously diluted. The mean fluorescence intensity (MFI) of the dye was also plotted with cell concentration over time to show the inverse relationship between the two. This type of assay is often used to look at changes in T lymphocyte activation.

The high sensitivity and high resolution flow cytometer are suitable for all kind of laboratories. The instrument provides a 1 to 3 laser option, exchangeable filters, and multiple sampling options. Together with the user-friendly software, this results in broad application options in the field of life science research. The instrument is able to analyse samples in single tubes, multi-tube racks, or 24-, 48- and 96-well plates. The customizable laser and the detection of up to 17 parameters, make the NovoCyte a very flexible instrument. The NovoCyte has enhanced usability with fixed optical alignment, 24-bit detection dynamic ranges and no need for PMT voltage adjustment, accurate volumetric-based cell counting, pressure sensors to monitor fluidic status in real-time and offers automatic cleaning and decontamination procedures.