Fibroblasts were harvested and washed two times with PBS, resuspended at a final concentration of 1 x 106 cells/ml, then stained with 5 [micro]mol/L CFSE
(Invitrogen, Carlsbad, CA, USA), and incubated at 37[degrees]C for 10 min.
Using flow cytometry to detect epithelial cell adhesion molecule positive ([EpCAM.sup.+]) EVs from MCF7 cell culture and integrin /33 positive (CD61) EVs from human plasma, we compared the performance of the most promising generic markers, namely, calcein AM, calcein violet, CFSE
, di-8-ANEPPS, and lactadherin, and used side scatter as a reference.
The Luminex IFNy and TNFa read-outs in combination with CFSE
staining (T cell proliferation) and CD69 expression (T cell activation) were subsequently used to investigate the specificity of the test substance-induced T cell response during earlier stages of co-culture, before re-stimulation.
Carboxyfluorescein diacetate succinimidyl ester (CFSE
) was purchased from Molecular Probes (Eugene, OR).
DCs were activated by LPS in the presence or absence of conditioned medium from the RM-1 as well as PG[E.sub.2] and stained with CFSE
. The percentage of CFSE-positive DCs migrating to the draining inguinal lymph nodes collected from mice injected with DCs was assessed by flow cytometry.
Labeling of CFSE
and Evaluation of Lymphocyte Proliferation
Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE
The in vitro lymphocyte proliferation (CFSE
assay) was not influenced by the BS extracts if cells were cultured without any activator (data not shown) or if stimulated by PHA.
Splitter, "Antigen specific in vivo killing assay using CFSE
labeled target cells," Journal of Visualized Experiments, no.
Mn(II) shows d5 electronic configuration which has no any crystal field stabilization energy (CFSE
) specific to any particular geometry.
DMSO, N-formyl-methionylleucyl-phenylalanine (fMLP), lipopolysaccharide (LPS), phorbol, 12-myristate 13-acetate, CFSE
, ammoniumpersulfate, bovine serum albumine, ethanol absolute, hydrochloric acid (37%), leupeptin hemisulfate, p-coumaric acid, pepstatin A, ponceau S, and trypan blue and Tween 20, and monoclonal antibodies against [beta]-actin were obtained from Sigma-Aldrich (Taufkirchen, Germany).
Briefly, 5(6)-carbosyfluorescein diacetate N-succinimidyl ester (CFSE
) labeled PBMCs are incubated with stimulants for four to five days at 37oC/5% CO2 and determine the number of fluorescent peaks corresponding to cell divisions of the parent cell population (Figure 2).