Technical Note
The Effect of Trypan Blue Treatment on Autofluorescence of Fixed Cells Olga N. Shilova,1 Evgeny S. Shilov,2 Sergey M. Deyev1,2*
1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, MiklukhoMaklaya St., 16/10, Moscow, 117997, Russian Federation
2
Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow, 119234
Received 28 December 2016; Revised 24 July 2017; Accepted 11 August 2017 Grant sponsor: Russian Science Foundation, Grant number: 14–24-00106; Grant sponsor: Russian Foundation for Basic Research, Grant number: 17-04-00308 Additional Supporting Information may be found in the online version of this article. *Correspondence to: Sergey M. Deyev, Miklukho-Maklaya St., 16/10, Moscow 117997, Russian Federation. E-mail:
[email protected] Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.23199 C 2017 International Society for V
Advancement of Cytometry
Cytometry Part A 00A: 0000, 2017
Abstract Controlling background fluorescence remains an important challenge in flow cytometry, as autofluorescence can interfere with the detection of chromophores. Furthermore, experimental procedures can also affect cellular fluorescence in certain regions of the emission spectrum. In this work, the effects of fixation, permeabilization, and heating on cellular autofluorescence are analyzed in various spectral regions, along with the influence of trypan blue as a quenching dye for these treatments. The impact of these procedures on the staining of SK-BR-3 cells with a dim green fluorophore, a miniSOG (mini Singlet Oxygen Generator) flavoprotein in the form of the recombinant protein DARPin-miniSOG, is also evaluated. The data presented here indicate that fixation of certain types of cells leads to noticeable increase of the autofluorescence. Our results also suggest that trypan blue should be used as an autofluorescence quencher only with bright green emitters since it interferes with the fluorescent signal in a longerwavelength region of the spectrum and as a result causes reduction of the signal from C 2017 International Society for Advancement of Cytometry V dim green fluorescent agents. Key terms autofluorescence; trypan blue; DARPin; miniSOG; flow cytometry
SPECIFIC fluorescent dyes and fluorescent protein expression are widely used in fundamental studies and clinical tests. The success in this field is substantial, but in many cases, autofluorescence or background fluorescence remains a problem (1). Although in flow cytometry, the term ‘autofluorescence’ is often referred to as background fluorescence excited by either ultraviolet irradiation or visible, violet or blue light and is characterized by emission in the green part of the spectrum, autofluorescence can also be observed at longer wavelengths. Some sources of background fluorescence in mammalian cells are summarized in Figure 1 (2–6). There may be specific sources of autofluorescence in certain cells types, such as retinols or melanin (2), but cellular molecules present in all types of cells can also increase background fluorescence. Additional sources of autofluorescence include oxidoreductase cofactors and coenzymes: nicotinamide nucleotides (emission in the range of 400–550 nm) and flavins (480–600 nm) (3,4). Lipofuscin and other lipopigments can emit 450 to 700 nm (3,5). Porphyrins, including heme and its derivatives, can emit in the range of 600–700 nm (6). Notably, some standard techniques such as formaldehyde fixation or thermocycling can increase the signal corresponding to the background fluorescence. To distinguish between autofluorescence and the signal of interest, a number of procedures have been developed. For fixed cells or tissues, one can use quenching dyes, including trypan blue (7), Eriochrome Black T or Sudan Black B (8). Another way to reduce autofluorescence of fixed samples both for flow cytometry and for histology is to treat them with reducing chemicals such as sodium borohydride (9,10). To date, a large variety of technical methods for avoiding autofluorescence in microscopy have been developed, ranging from bleaching a sample with bright light before staining
Technical Note
Figure 1. Sources of autofluorescence in mammalian cells. [Color figure can be viewed at wileyonlinelibrary.com]
(11) to sophisticated methods such as confocal microscopy, multiphoton excitation, and deconvolution techniques (12). Nevertheless, in flow cytometry, appropriate sample preparation provides a major contribution to controlling autofluorescence. In this article, we studied the effects of trypan blue staining on cellular autofluorescence of intact cells, cells fixed with 4% formaldehyde, cells fixed with 4% formaldehyde and permeabilized with Tween 20, as well as cells fixed using ethanol. Fixation is one of possible causes of autofluorescence intensity change. The increase in fluorescence after aldehyde fixation is considered to be common and is thought to be caused by Schiff acid-base reactions with amine groups generating adducts that are intensely fluorescent (13). Formaldehyde might also be involved in lipopigment formation, leading to increased autofluorescence. Another commonly used fixative is ethanol. The literature concerning the influence of ethanol fixation on cell autofluorescence is rather poor, and considers ethanol to be a good fixative with minimal effect on background fluorescence (14). We also analyzed heating as a factor increasing autofluorescence during the processes of thermocycling or paraffin embedding. We used the breast adenocarcinoma cell line SK-BR-3, an immortalized CHO cell line and primary murine thymocytes. The effect of trypan blue as a quenching agent for green fluorescence was also examined in the case of green fluorescent dye. As a model of a problematic system, we used SK-BR-3 cells, which have relatively high autofluorescence, stained with the dim green fluorophore DARPin-miniSOG. This recombinant protein was originally designed as a targeted photosensitizer miniSOG (15) specifically binding to the receptor ErbB2 (HER2) on the cell surface because of DARPin_9-29 module (16). The fluorescent properties of DARPin-miniSOG are provided by flavin 2
mononucleotide (FMN) cofactor of miniSOG. Its fluorescence is detectable by a flow cytometer (17), though the intensity is relatively low.
MATERIALS AND METHODS Sample Preparation Cultivated cells were grown in complete McCoy’s 5 A media (HyClone Laboratories, Logan, Utah, USA) supplemented with 10% fetal bovine serum (GE Healthcare; Logan, Utah, USA) at 378C in a humidified incubator with 5% CO2. The mice for thymocytes isolation were purchased from the SPF (specified pathogen-free) licensed nursery of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences. The procedures were conducted according to the protocol approved by the Bioethics Committee of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences. C57BL/6 mice aged 12–15 weeks were euthanized by cervical dislocation; then, the thymus was removed, and thymocytes were resuspended in phosphate buffered saline (PBS, PanEco; Moscow, Russian Federation) and filtered through 70 lm filters (BD Falcon; Bedford, Massachusetts, USA). Fixation and Permeabilization Cultivated cells were detached from plastic using sterile Versen solution (PanEco). The cell suspension was washed with PBS and used in further experiments, 1–2 3 105 cells were used for each sample. During all manipulations, cells were kept on ice, and formaldehyde (AppliChem GmbH; Darmstadt, Germany), Tween-20 (SERVA Electrophoresis GmbH; Heidelberg, Germany), and trypan blue (PanEco) solutions were made in PBS. Autofluorescence and Trypan Blue in Fixed Cells
Technical Note For cell fixation, we used two standard reagents: ethanol (Ferane; Moscow, Russian Federation) and formaldehyde. For ethanol fixation, cells were centrifuged at 800 g for 4 min, the supernatant was removed, and the cell pellet was resuspended in 200 ll of cold 96% ethanol, incubated 15 min on ice, washed with PBS to remove the ethanol, and resuspended in PBS. For formaldehyde fixation, the cell pellet was resuspended in 100 ll of 4% phosphate-buffered formaldehyde, incubated for 10 min on ice, and washed with PBS. For permeabilization, after washing, formaldehyde-fixed cells were resuspended in 100 ll of 0.2% Tween-20 solution, incubated for 30 min on ice, and then washed with PBS. Heating was performed using a Thermomixer comfort thermostat (Eppendorf; Hamburg, Germany): cells were resuspended in 500 ll of PBS and incubated at 758C for 10 min. In each experiment the treated samples were divided into two groups. In one group, autofluorescence was measured immediately; in the second, cells were treated with trypan blue and then analyzed. For trypan blue staining, the samples were incubated in 0.4% trypan blue solution for 10 min on ice and washed twice with PBS. All experiments were repeated 3 to 6 times. Expression and Purification of DARPin-miniSOG The protein was produced in cytoplasm of E. coli and purified using affine chromatography. The construction of the plasmid encoding DARPin-miniSOG and the expression and purification procedures were previously described in detail (16). Briefly, the coding sequence of miniSOG was cloned into the vector pET-22 b in the same reading frame as the DARPin-9_29 coding sequence. The protein was expressed in Escherichia coli BL21(DE3) cells in a soluble form in the cytoplasm and was purified using Ni21-NTA columns (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) according to the manufacturer’s instructions. The DARPin protein was produced the same way, conjugated with fluorescein isothiocyanate (FITC, Sigma-Aldrich, St. Louis, Missouri, USA), and purified from excess amount of FITC using Zeba spin desalting column (Thermo Scientific, Rockford, MA, USA) according to the manufacturer’s instructions. Staining of SK-BR-3 Cells with DARPin-miniSOG SK-BR-3 cells were stained with DARPin-miniSOG before any other treatments. Samples of SK-BR-3 cells were incubated in PBS containing 1 lM DARPin-miniSOG for 10 min on ice and washed once with PBS. Then, one sample was left as a control, one was stained with trypan blue, and the others underwent fixation, permeabilization and trypan blue staining as described above. During all manipulations, cells were kept on ice to prevent possible internalization of DARPin-miniSOG in complex with ErbB2 before fixation, as that can decrease the fluorescence intensity of miniSOG. DARPin-FITC was also used as a 1 lM solution according to the same protocol. Flow Cytometry Details Background fluorescence was measured using a Gallios flow cytometer (Beckman Coulter; Brea, California, USA) equipped with three lasers: 488 nm (22 mW), 638 nm Cytometry Part A 00A: 0000, 2017
Table 1. Settings used in the Gallios flow cytometer
LASER
CHANNEL
488 nm
FS SS FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 FL9 FL10
638 nm
405 nm
VOLTAGE, V
FILTER FOR FLUORESCENCE DETECTION
296 502 478 469 478 604 615 666 601 545 357 376
– – 525/40 BP 575/30 BP 620/30 BP 695/30 BP 755 LP 660/20 BP 660/20 BP 755 LP 450/40 BP 550/40 BP
(25 mW) and 405 nm (40 mW). The corresponding filters and voltages are summarized in Table 1. Channels FL1: 525/ 40 nm, FL2: 575/30 nm, FL3: 620/30 nm, FL4: 695/30 nm and FL5: 755 nm LP corresponded to a blue (488 nm) laser. Channels FL6: 660/20 nm, FL7: 725/20 nm and FL8:755 nm LP corresponded to a red (638 nm) laser. Light emitted by a violet (405 nm) laser was detected in using the channels FL9: 450/ 40 nm and FL10: 550/40 nm. The events were gated using two step gating strategy: gated for cells according to forward and side scattering (FSC-Area/SSC-Area) in a linear scale and then subsequently gated for single cells on a FSC-Width/FSC-Area plot. The gating strategy is shown in Figure 2. All the data concerning fluorescence intensity was analyzed in logarithmic scale. The data were processed using Gallios software. DARPin-miniSOG staining was analyzed on a BD Accuri C6 flow cytometer (Becton Dickinson, Ann Arbor, Michigan, USA). Green fluorescence was excited using a blue (488 nm) laser (20 mW) and analyzed in the channel FL1: 533/30. The acquired data were analyzed with BD Accuri C6 Software. Files are available upon request from the corresponding author. Cell Size Estimation Cell sizes were estimated using a Leica DMI6000B microscope (Leica Microsystems CMS GmbH; Wetzlar, Germany). Detached SK-BR-3 and CHO cells and thymocytes were placed in 24-well plates in PBS and imaged at x400 magnification. Cell sizes were estimated using Leica Application Suite software. Trypan Blue Spectrum Analysis Trypan blue and flavin mononucleotide spectra and fluorescence intensities were analyzed using Infinite M1000 PRO spectra analyzer (Tecan; Mannedorf, Switzerland). Bovine serum albumin (BSA, Merck; Darmstadt, Germany) was used as a 1% (m/m) solution in PBS (PanEco), trypan blue was used as a 0.4% solution either in PBS or in PBS containing 1% BSA. Absorbance spectra of BSA and trypan blue were analyzed in range 230–620 nm in Greiner 384-well transparent plates, emission spectra were analyzed in range 500–850 nm, emission was excited with 480 nm light (480/5 BP). Flavin 3
Technical Note
Figure 2. Gating strategy for SK-BR-3 cells. A: Common strategy used for gating on singe events. B: Gating for cells after the treatments. Cells stained with trypan blue are shown in the right panel. [Color figure can be viewed at wileyonlinelibrary.com]
mononucleotide (FMN, Pharmstandard; Moscow, Russian Federation) was used as 5 mg/mL solution in PBS containing 1% BSA. FMN absorption spectrum was analyzed in range 230–550 nm, FMN emission was excited with 400 nm light (400/5 BP) and emission spectrum was analyzed in range 420–700 nm. To evaluate the effect of trypan blue on FMN 4
fluorescence we compared fluorescence intensities of FMN in PBS containing 1% BSA with that of FMN solution containing 1% BSA and 0.4% of trypan blue. The fluorescence was excited by blue light (488/5 nm BP) and analyzed in range referring to FL1 channel (535/20 BP). The data was analyzed with Magellan 7.2 software. Autofluorescence and Trypan Blue in Fixed Cells
Technical Note Data Analysis Flow cytometry data were analyzed using FlowJo software. Autofluorescence intensity is expressed as the mean log fluorescence intensity of a single cell population in the corresponding detection channel. Staining indexes for SK-BR-3 cells treated with DARPin-miniSOG were calculated according to the following formula (17):
SI5
meanðpositiveÞ2meanðnegativeÞ 2 SDðnegativeÞ
Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). Comparison between results was performed using the Mann-Whitney and Kolmogorov-Smirnov tests. Statistical significance was defined as P < 0.05.
RESULTS AND DISCUSSION Fluorescence of Intact Cells In most studies involving fluorescence detection, high background fluorescence can become a problem when blue, violet, or ultraviolet light is used for excitation, as the irradiation can also excite a broad spectrum of natural cellular fluorophores. For comparing fluorescence levels, we used different mammalian cells: primary obtained murine thymocytes, stably cultivated SK-BR-3 (human breast adenocarcinoma) and CHO (Chinese hamster ovary) cell lines. The highest level of autofluorescence in FL1, FL2, FL9 and FL10 was observed in SK-BR-3 cells, the fluorescence levels were lower in CHO cells, and the lowest levels were observed for thymocytes. For example, the mean fluorescence intensities (MFI) in FL1 channel for SK-BR-3 cells reached 7790 6 536 units, while for CHO it was 3341 6 363 units, for thymocytes it was 394 6 6 units (all raw data are shown in supplementary material). This difference can be a consequence of differences in cell size that result in different amounts of natural fluorophore groups per cell. According to the microscopic data, the average diameters of SK-BR-3 cells, CHO cells and thymocytes were equal to 16 6 2.3, 12 6 2.4, and 6 6 0.8 um respectively (data are shown as the mean 6 SEM). To decrease the contribution of cell size in autofluorescence differences, we normalized the mean fluorescence intensities to the average cell size of 100 lm2. The size-normalized data for the channels FL-1, FL-2, FL-9 and FL-10 are summarized in Figure 3. After normalization the cells show comparable levels of basic autofluorescence, with two exceptions: SK-BR-3 cells demonstrate relatively high fluorescence in FL9 channel and thymocytes demonstrate relatively high fluorescence in FL2 channel. The Influence of Fixation, Permeabilization on Label-Free Cells Mean fluorescence intensity data for channels corresponding to short wavelength emission are summarized in Figure 3. As shown in the plots, the level of autofluorescence depends on the fixation protocol as well as on the cell line used. Green fluorescence excited by a blue (488 nm) laser was analyzed in the channel FL1 (525/40 BP). Most of the used experimental techniques did not significantly affect green Cytometry Part A 00A: 0000, 2017
autofluorescence, though ethanol fixation decreased the background fluorescence in SK-BR-3 cells, and heating led to increased autofluorescence in thymocytes. The effect of ethanol in FL1 may be associated with the extraction of lipopigments (18). Yellow fluorescence excited by the blue laser was analyzed in the channel FL2 (575/30 BP). In channel FL2, heating and ethanol fixation of thymocytes led to increased fluorescence, although ethanol fixation of CHO cells decreases FL2 autofluorescence. Blue fluorescence excited by a violet (405 nm) laser was analyzed in channel FL9 (450/40 BP). In this channel, the most dramatic autofluorescence changes occurred in SK-BR-3 cells, in which all the tested fixation and permeabilization techniques caused significant decreases in autofluorescence. The FL10 channel (550/40 BP) was used to analyze green fluorescence excited by the violet laser. In this channel, no significant increase in autofluorescence because of experimental techniques was observed. The other six channels were withdrawn from this consideration because of extremely high fluorescence of trypan blue treated cells in red part of spectrum, excluding any chances for another fluorophore usage in these channels (see the data for all channels in the Supplementary materials). The Effect of Trypan Blue Treatment on Autofluorescence of Label-Free Cells Trypan blue was used in the analysis as one of the standard chemicals advised for short wavelength autofluorescence removal for flow cytometry (19). Based on its emission spectrum (7), it should be useful for quenching green fluorescence and as a reporting dye, given that it can be used for distinguishing live and dead cells (20). Our data about trypan blue influence on cellular autofluorescence are demonstrated in Figure 3. Trypan blue treatment have controversial effect on cells, increasing autofluorescence in FL1 channel in case of living SK-BR-3 (about 50%) and decreasing autofluorescence for fixed/permeabilized SK-BR-3 and CHO, heated SK-BR-3, and ethanol treated CHO. Trypan blue have no effect for all types of treated thymocytes and for formalin fixed SK-BR-3 and CHO without permeabilization. The autofluorescence reduction in SK-BR-3 and CHO cells caused by trypan blue is modest in case of fixation/permeabilization (about 25%–40%) and more efficient in case of heating (about 60%). Trypan blue increased the cellular autofluorescence in FL2 channel in many cases including all types of formaldehyde fixed and fixed/permeabilized cells, living SK-BR-3 and thymocytes, and ethanol treated thymocytes because of its own emission. In these cases yellow fluorescence increasing varied from 13% on living thymocytes to 104% on fixed/permeabilized SK-BR-3. We therefore conclude that the use of trypan blue is not appropriate with yellow fluorescent dyes. Trypan blue treatments have no significant influence on autofluorescence in FL9 and FL10 channels in all tested cases. Our results demonstrate that trypan blue reduces the autofluorescence level of only fixed cells and might cause an opposite effect on the fluorescence of alive ones; the autofluorescence of SK-BR-3 cells significantly increased in FL1 5
Technical Note
Figure 3. Mean size-normalized autofluorescence levels of tested cells in channels referring to short wavelength excitation and emission. In channels FL1 and FL2, fluorescence excited by a blue (488 nm) laser is registered using 525/40 and 575/30 bandpass filters, respectively; in channels FL9 and FL10, fluorescence excited by a violet (405 nm) laser is registered using 450/40 and 550/40 bandpass filters, respectively. Error bars represent the standard error of the mean (SEM). Significant differences are marked with a star. The bars between the columns indicate samples that significantly differ from the untreated control. Untr – untreated cells, Form – formaldehyde treatment, Form 1 Tw20 – formaldehyde treatment followed by Tween-20 treatment, Heat – heating, EtOH – ethanol fixation.
and FL2 channels upon addition of trypan blue, as well as autofluorescence of thymocytes in FL2 channel. We therefore advice use of trypan blue to reduce the autofluorescence of 6
only fixed cells. For most experimental treatments, trypan blue decreased FL1 autofluorescence of fixed cells, with exceptions for SK-BR-3 cells and thymocytes fixed with ethanol and Autofluorescence and Trypan Blue in Fixed Cells
Technical Note thymocytes fixed with heating, which demonstrated insignificant increases in autofluorescence. In channel FL2, trypan blue led to the fluorescence increase because the 575/30 BP filter did not cut off its fluorescence. Regarding fluorescence excited with the violet (405 nm) laser, we can conclude that trypan blue did not cause significant changes in autofluorescence.
Figure 4. Staining indexes for SK-BR-3 cells with or without treatment with TB. The SIs are shown as the mean 6 SEM. Stars indicate significant difference caused by trypan blue.
Fluorescence of SK-BR-3 Cells Stained with DARPin-miniSOG Autofluorescence increase is usually harmful in case of dim fluorophores. For modeling this situation we stained SKBR-3 cells with the recombinant ErbB2-specific protein DARPin-miniSOG. This protein binds to ErbB2 molecule that is abundantly present on the surface of SK-BR-3 cells because of DARPin_9–29, the targeting module of nonimmunoglobulin origin. The fluorescent properties of DARPin-miniSOG are based on flavin mononucleotide (FMN) cofactor of miniSOG that provides green fluorescence excited by blue light, though the fluorescence intensity is relatively low. For example, the mean fluorescence intensity of SK-BR-3 cells stained with 1 lM DARPin-miniSOG in FL1
Figure 5. A: Absorbance spectra of bovine serum albumin, trypan blue and bovine serum albumin and trypan blue together. BSA - 1% bovine serum albumin; TB - 0.4% trypan blue; BSA 1 TB - % bovine serum albumin 0.4% trypan blue. B: Emission spectra of bovine serum albumin, trypan blue and bovine serum albumin and trypan blue together. BSA - 1% bovine serum albumin; TB - 0.4% trypan blue; BSA 1 TB - 1% bovine serum albumin 1 0.4% trypan blue. C: Absorbance and emission spectra of flavin mononucleotide and absorbance spectrum of trypan blue in presence of 1% bovine serum albumin. FMN excitation — the excitation spectrum of flavin mononucleotide; FMN emission — the emission spectrum of flavin mononucleotide; BSA 1 TB excitation - the absorbance spectrum of 1% bovine serum albumin 1 0.4% trypan blue. D: The intensity of flavin mononucleotide green fluorescence (535/20 bandpass filter) with and without trypan blue. FMN 1 BSA - fluorescence intensity of flavin mononucleotide in presence of 1% bovine serum albumin; FMN 1 BSA 1 TB - fluorescence intensity of flavin mononucleotide in presence of 1% bovine serum albumin and 0.4% trypan blue. [Color figure can be viewed at wileyonlinelibrary.com]
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Technical Note channel corresponding to green fluorescence excited by 488 nm laser (FL1: 533/30 nm bandpass filter) was 47930 6 4024, and for SK-BR-3 cells stained with 1 lM DARPin conjugated with FITC the mean fluorescence intensity was 1,06,720 6 15,790 (the data were recorded in logarithmic scale mode and represent mean 6 SEM). To compare the results of the staining we used staining index, the parameter that reflects both the increase of fluorescence intensity and resolution of stained and untreated populations. The staining results are presented as staining indexes (SIs) in Figure 4. We can conclude than none of the tested fixation or permeabilization methods reduced staining efficiency, with the exception of heating, which must have caused complete denaturation of miniSOG protein and loss of the FMN cofactor. Change in Fluorescence of SK-BR-3 Cells Stained with DARPin-miniSOG after Trypan Blue Treatment To investigate whether the use of trypan blue can improve the staining of cells with dim green dye due to quenching of background fluorescence, we applied it for treating SK-BR-3 cells stained with the recombinant DARPinminiSOG. The staining results are presented as staining indexes (SIs) in Figure 4. The treatment of cells with 0.4% trypan blue did not improve staining and instead made the staining poorer. Thus, the use of trypan blue did not improve the resolution of stained and unstained populations, and trypan blue should not be used with dim fluorophores, at least at these concentrations. Trypan Blue Reduces Fluorescence of Flavin Nucleotides In Vitro Trypan blue action is known to be based on quenching: it can absorb light emitted by the inner cellular sources (21). To verify the mechanism of trypan blue action on autofluorescence we measured fluorescence and absorption spectra in pure solution of trypan blue in PBS and in a mixture with bovine serum albumin. As a negative control we used PBS with the same concentration of BSA. Trypan blue was previously shown to nonspecifically bind BSA (22) and other proteins with no regard to amino acid residues sequence (23). The light absorption of trypan blue increases in the presence of protein (Fig. 5A), which is in agreement with the previously published data (20). As an example of important source of cellular autofluorescence we used flavin mononucleotide, a usual cofactor of oxidoreductases which was shown to make significant contribution in green background fluorescence of mammalian cells (4). According to FMN emission spectrum shown in Figure 5C trypan blue is likely to absorb the light emitted by FMN which is supported by the decrease of FMN fluorescence intensity in the presence of trypan blue and BSA (Fig. 5D). Nevertheless, the emission spectrum of trypan blue does not favor its use with dyes emitting in yellow part of the spectrum and further. The emission spectrum of trypan blue in presence of BSA shifts to the short wavelength side, so the range of detectable fluorescence of trypan blue begins with wavelength 585 nm instead 600 nm (Fig. 5B, fluorescent 8
spectra shown in logarithmic scale). This spectral change can explain increase of cellular fluorescence in FL2 channel after trypan blue treatment. Addition of trypan blue to complex system completely diminished fluorescence of proteins (e.g., BSA), modestly decreased fluorescence of nucleotides (e.g. FMN) and caused fluorescence of trypan blue itself, which can be detected in FL2 channel after spectral shift.
CONCLUSION In general, the observed effects of fixation on the autofluorescence depend strongly on the cell type. Our results support the use of trypan blue as a quenching agent for green autofluorescence excited by blue light for fixed cells in case of cell membrane permeabilization. In case of green autofluorescence excited by violet light trypan blue treatment does not decrease background fluorescence significantly. In the case of dim fluorescent dyes such as DARPinminiSOG, trypan blue fluorescence treatment can interfere with the measured signal and decrease the staining index, so its usage should be carefully analyzed in each case.
ACKNOWLEDGMENTS DARPin-miniSOG production and purification were supported by the Russian Science Foundation (grant 14-2400106). Cell culture studies were supported by the Russian Foundation for Basic Research (grant 17-04-00308). The authors are grateful to Galina Proshkina and Victoria Shipunova from Laboratory of Molecular Immunology of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences for DARPin-miniSOG purification and manuscript revision. The experiments were performed using equipment purchased through the Lomonosov Moscow State University Program of Scientific Development.
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