Autofluorescence of cells

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

Roshchina V.V. (2003). Autofluorescence of plant secreting cells as a biosensor and bioindicator reaction. Journal of Fluorescence 13 403-420. 

Reduction of cell autofluorescence relative to fluorescence excited at cell/substrate contacts. 

Aubin, J. E. (1979) Autofluorescence of viable cultured mammalian cells. J. Histo-chem. Cytochem. 27, 36-43. 

Fig. 9. Flow analysis of apoptotic human peripheral blood lymphocytes using direct TUNEL assay. Human peripheral blood lymphocytes (1 x 10 ) treated (A) without or (B) with dexamethasone (0.1 ijlM) for 16 h were transferred to a 15-ml tube. Paraformaldehyde (2%) was added to cells with shaking and incubated for 10-15 min in ice with occasional shaking. Cells were washed with PBS with 1% BSA and 3-4 ml cold acetone was then added to cells. After 2-3 min incubation on ice with occasional shaking, cells were washed twice and TUNEL reaction mixture including enzyme TdT and fluorescein-labeled anti-dUTP antibody was added to cells (H). For the negative control group, only label solution without TdT was added to cells ( ). Cells without any addition of reaction or label solution were used for assessment of the autofluorescence ( ). The cell mixture was incubated 1 h at 37°C in the dark. The result of the apoptosis after flow analysis was expressed as a histogram using software CellQuest (Becton Dickinson). In Fig. 9A Ml (nonapoptotic cells), 86% M2 (apoptotic cells), 14%. In Fig. 9B Ml (nonapoptotic cells), 78% M2 (apoptotic cells), 22% (our unpublished data).

Cells may show a low level of autofluorescence at 413 nm when irradiated at 324 nm. This fluorescence dramatically increases when d -parinaric acid (159) is incorporated into the cell membrane, either by intercalation or esteriflcation. Exposure to oxidation stress of cells enriched with the 159 fluorescent probe causes diminution of the fluorescence intensity and is directly correlated with formation of lipid hydroperoxides. Addition of antioxidants, such as Vitamin E (21), abates fluorescence diminution. A blanc run of cells enriched with 159 but not subjected to oxidation stress is necessary to follow the degradation of 159 when exposed to UV irradiation. This method was applied to track lipid oxidation during apoptosis and other phenomena, triggered by toxic compounds such as H2O2, f-BuOOH and cumyl hydroperoxide (27)"° 11,424... 

It has been reported that incubation of cells in 0.1% sodium borohydride in 1 mL PBS for 30 min at room temperature, after the antibody incubations, reduces autofluorescence and nonspecific staining This has not proved to be advantageous in the author s hands... 

The resulting determination of the numbers of antireceptor antibody molecules bound/epithelial cell are shown in Table 2. The value for the control antibody represents the autofluorescence of the target cells in terms of the equivalent number of fluorescent-labeled antibody molecules, rather than binding of the control antibody. Thus, a similar value was obtained for target cells with and without control antibody (not shown). 

Fig. 10.6. Detection of rare fetal erythrocytes in the maternal circulation. The fetal erythrocytes are HbF positive and low in autofluorescence. The cells in R1 are displayed and enumerated in the bottom right histogram. From Davis (1998).

Fig. 11.6. Flow cytometric analysis of surface water from points at 1.5 mile intervals off shore from Cape Flatteras, North Carolina. Forward scatter and orange autofluorescence identify two Synechococcus populations with different phycoerythrin content. Beads were used to calibrate the number of cells present. From Chisholm et al. (1986).

There may well be increasing interest in analysis of the intrinsic characteristics of cells that lead to alterations in autofluorescence. John Steinkamp, Harry Crissman, and others at the Los Alamos Laboratory have been using flow systems to study the time character-... 

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

---