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Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.

Lin JR, Fallahi-Sichani M, Sorger PK - Nat Commun (2015)

Bottom Line: Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers.Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited.Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.

View Article: PubMed Central - PubMed

Affiliation: HMS LINCS Center &Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 USA.

ABSTRACT
Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.

No MeSH data available.


Related in: MedlinePlus

Multiplexed imaging of single-cell using Cyclic ImmunoFluorescence (CycIF).(a) An overview of the CycIF procedure. Four-colour staining alternates with fluorophore inactivation by oxidation to progressively build a multichannel image. (b) CycIF procedure using direct immunofluorescence (with fluorophore-conjugated antibodies) and chemical inactivation of fluorophores. COLO858 melanoma cells were fixed and stained using antibodies for Ki-67 (Alexa 488), p-Histone H3 (Alexa 555), p21 (Alexa 647) and Hoechst (left panel). Cells were exposed to fluorophore-inactivation by oxidation using hydrogen peroxide, high pH and light and then reimaged (middle panel) to confirm efficient bleaching. Cells were then stained with fluorophore-conjugated antibodies for p-S6S240/244 (Alexa 488), p-RbS807/811 (Alexa 555), p-S6S235/236 (Alexa 647) and Hoechst. (c) CycIF procedure using indirect immunofluorescence and protease-mediated antibody stripping. MCF7 cells were fixed and stained using primary antibodies for p-ERK1/2T202/Y204 (rabbit), p53 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (left panel). Cells were digested with pepsin/papain mixture (see Methods for details) and reimaged (middle panel). Cells were restained using primary antibodies for p-RbS807/811 (rabbit), p-Histone H2A.XS139 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (right panel). (d) Bleaching rate for Alexa 488, 555 or 647-conjugated antibodies following incubation in a base-hydrogen peroxide mixture. (e) Correlation of signal intensities after using the same antibodies in successive CycIF cycles. Five-cycle CycIF was applied to COLO858 cells treated with increasing doses of vemurafenib (error bars show the range of biological duplicates). Cells were stained with Alexa 488-conjugated p-ERK antibody, and p-ERK signal intensities from different CycIF rounds were quantified and compared. Cells (1,000–2,000) were imaged for each condition and well mean intensity values across duplicates were reported. Error bars indicate s.d. (f) Quantification of cell loss based on Hoechst staining (averaged across n=30 different wells) through cycles of CycIF. Cell numbers from each well after each CycIF cycle were normalized to the mean cell number derived from cycle 1 and presented in box-and-whisker plots with mean values (shown by red lines), interquartile ranges (shown as boxes) and whiskers (representing the 1st/99th percentiles).
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f1: Multiplexed imaging of single-cell using Cyclic ImmunoFluorescence (CycIF).(a) An overview of the CycIF procedure. Four-colour staining alternates with fluorophore inactivation by oxidation to progressively build a multichannel image. (b) CycIF procedure using direct immunofluorescence (with fluorophore-conjugated antibodies) and chemical inactivation of fluorophores. COLO858 melanoma cells were fixed and stained using antibodies for Ki-67 (Alexa 488), p-Histone H3 (Alexa 555), p21 (Alexa 647) and Hoechst (left panel). Cells were exposed to fluorophore-inactivation by oxidation using hydrogen peroxide, high pH and light and then reimaged (middle panel) to confirm efficient bleaching. Cells were then stained with fluorophore-conjugated antibodies for p-S6S240/244 (Alexa 488), p-RbS807/811 (Alexa 555), p-S6S235/236 (Alexa 647) and Hoechst. (c) CycIF procedure using indirect immunofluorescence and protease-mediated antibody stripping. MCF7 cells were fixed and stained using primary antibodies for p-ERK1/2T202/Y204 (rabbit), p53 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (left panel). Cells were digested with pepsin/papain mixture (see Methods for details) and reimaged (middle panel). Cells were restained using primary antibodies for p-RbS807/811 (rabbit), p-Histone H2A.XS139 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (right panel). (d) Bleaching rate for Alexa 488, 555 or 647-conjugated antibodies following incubation in a base-hydrogen peroxide mixture. (e) Correlation of signal intensities after using the same antibodies in successive CycIF cycles. Five-cycle CycIF was applied to COLO858 cells treated with increasing doses of vemurafenib (error bars show the range of biological duplicates). Cells were stained with Alexa 488-conjugated p-ERK antibody, and p-ERK signal intensities from different CycIF rounds were quantified and compared. Cells (1,000–2,000) were imaged for each condition and well mean intensity values across duplicates were reported. Error bars indicate s.d. (f) Quantification of cell loss based on Hoechst staining (averaged across n=30 different wells) through cycles of CycIF. Cell numbers from each well after each CycIF cycle were normalized to the mean cell number derived from cycle 1 and presented in box-and-whisker plots with mean values (shown by red lines), interquartile ranges (shown as boxes) and whiskers (representing the 1st/99th percentiles).

Mentions: CycIF has several variants (Supplementary Fig. 1), but the most generally useful involves three-channel IF using antibodies that are chemically coupled to one of three different Alexa dyes, while the fourth channel is used for Hoechst 33342 to visualize DNA and count cells. Fluorophores are then inactivated by oxidation using hydrogen peroxide24, high pH and light, followed by a wash step and a second round of staining and imaging (Fig. 1a,b, Supplementary Fig. 2). Indirect-IF can also be used, but since the primary antibodies remain bound to their antigens, the number of channels is limited to the number of different species (rabbit, rat, goat and so on) of primary antibody and thus the number of unique secondary antibodies. A second CycIF variant enables multiple rounds of indirect-IF by using proteases such as papain or pepsin to digest primary and secondary antibodies (Fig. 1c, Supplementary Fig. 3); this protocol is a variation on ‘epitope recovery' techniques25. In a third CycIF variant, cells carrying FPs and other live-cell reporters are imaged and then bleached using low pH and hydrogen peroxide, followed by rounds of direct and indirect immunofluorescence. The use of a common dye in all cycles (Hoechst 33342 in the current work) makes it possible to assay for cell loss and register successive images at sub-pixel resolution (Fig. 1b; Hoechst is partially bleached by oxidation and we typically readd it at each cycle).


Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.

Lin JR, Fallahi-Sichani M, Sorger PK - Nat Commun (2015)

Multiplexed imaging of single-cell using Cyclic ImmunoFluorescence (CycIF).(a) An overview of the CycIF procedure. Four-colour staining alternates with fluorophore inactivation by oxidation to progressively build a multichannel image. (b) CycIF procedure using direct immunofluorescence (with fluorophore-conjugated antibodies) and chemical inactivation of fluorophores. COLO858 melanoma cells were fixed and stained using antibodies for Ki-67 (Alexa 488), p-Histone H3 (Alexa 555), p21 (Alexa 647) and Hoechst (left panel). Cells were exposed to fluorophore-inactivation by oxidation using hydrogen peroxide, high pH and light and then reimaged (middle panel) to confirm efficient bleaching. Cells were then stained with fluorophore-conjugated antibodies for p-S6S240/244 (Alexa 488), p-RbS807/811 (Alexa 555), p-S6S235/236 (Alexa 647) and Hoechst. (c) CycIF procedure using indirect immunofluorescence and protease-mediated antibody stripping. MCF7 cells were fixed and stained using primary antibodies for p-ERK1/2T202/Y204 (rabbit), p53 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (left panel). Cells were digested with pepsin/papain mixture (see Methods for details) and reimaged (middle panel). Cells were restained using primary antibodies for p-RbS807/811 (rabbit), p-Histone H2A.XS139 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (right panel). (d) Bleaching rate for Alexa 488, 555 or 647-conjugated antibodies following incubation in a base-hydrogen peroxide mixture. (e) Correlation of signal intensities after using the same antibodies in successive CycIF cycles. Five-cycle CycIF was applied to COLO858 cells treated with increasing doses of vemurafenib (error bars show the range of biological duplicates). Cells were stained with Alexa 488-conjugated p-ERK antibody, and p-ERK signal intensities from different CycIF rounds were quantified and compared. Cells (1,000–2,000) were imaged for each condition and well mean intensity values across duplicates were reported. Error bars indicate s.d. (f) Quantification of cell loss based on Hoechst staining (averaged across n=30 different wells) through cycles of CycIF. Cell numbers from each well after each CycIF cycle were normalized to the mean cell number derived from cycle 1 and presented in box-and-whisker plots with mean values (shown by red lines), interquartile ranges (shown as boxes) and whiskers (representing the 1st/99th percentiles).
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587398&req=5

f1: Multiplexed imaging of single-cell using Cyclic ImmunoFluorescence (CycIF).(a) An overview of the CycIF procedure. Four-colour staining alternates with fluorophore inactivation by oxidation to progressively build a multichannel image. (b) CycIF procedure using direct immunofluorescence (with fluorophore-conjugated antibodies) and chemical inactivation of fluorophores. COLO858 melanoma cells were fixed and stained using antibodies for Ki-67 (Alexa 488), p-Histone H3 (Alexa 555), p21 (Alexa 647) and Hoechst (left panel). Cells were exposed to fluorophore-inactivation by oxidation using hydrogen peroxide, high pH and light and then reimaged (middle panel) to confirm efficient bleaching. Cells were then stained with fluorophore-conjugated antibodies for p-S6S240/244 (Alexa 488), p-RbS807/811 (Alexa 555), p-S6S235/236 (Alexa 647) and Hoechst. (c) CycIF procedure using indirect immunofluorescence and protease-mediated antibody stripping. MCF7 cells were fixed and stained using primary antibodies for p-ERK1/2T202/Y204 (rabbit), p53 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (left panel). Cells were digested with pepsin/papain mixture (see Methods for details) and reimaged (middle panel). Cells were restained using primary antibodies for p-RbS807/811 (rabbit), p-Histone H2A.XS139 (mouse), Alexa 488-conjugated anti-rabbit, and Alexa 647-conjugated anti-mouse secondary antibodies (right panel). (d) Bleaching rate for Alexa 488, 555 or 647-conjugated antibodies following incubation in a base-hydrogen peroxide mixture. (e) Correlation of signal intensities after using the same antibodies in successive CycIF cycles. Five-cycle CycIF was applied to COLO858 cells treated with increasing doses of vemurafenib (error bars show the range of biological duplicates). Cells were stained with Alexa 488-conjugated p-ERK antibody, and p-ERK signal intensities from different CycIF rounds were quantified and compared. Cells (1,000–2,000) were imaged for each condition and well mean intensity values across duplicates were reported. Error bars indicate s.d. (f) Quantification of cell loss based on Hoechst staining (averaged across n=30 different wells) through cycles of CycIF. Cell numbers from each well after each CycIF cycle were normalized to the mean cell number derived from cycle 1 and presented in box-and-whisker plots with mean values (shown by red lines), interquartile ranges (shown as boxes) and whiskers (representing the 1st/99th percentiles).
Mentions: CycIF has several variants (Supplementary Fig. 1), but the most generally useful involves three-channel IF using antibodies that are chemically coupled to one of three different Alexa dyes, while the fourth channel is used for Hoechst 33342 to visualize DNA and count cells. Fluorophores are then inactivated by oxidation using hydrogen peroxide24, high pH and light, followed by a wash step and a second round of staining and imaging (Fig. 1a,b, Supplementary Fig. 2). Indirect-IF can also be used, but since the primary antibodies remain bound to their antigens, the number of channels is limited to the number of different species (rabbit, rat, goat and so on) of primary antibody and thus the number of unique secondary antibodies. A second CycIF variant enables multiple rounds of indirect-IF by using proteases such as papain or pepsin to digest primary and secondary antibodies (Fig. 1c, Supplementary Fig. 3); this protocol is a variation on ‘epitope recovery' techniques25. In a third CycIF variant, cells carrying FPs and other live-cell reporters are imaged and then bleached using low pH and hydrogen peroxide, followed by rounds of direct and indirect immunofluorescence. The use of a common dye in all cycles (Hoechst 33342 in the current work) makes it possible to assay for cell loss and register successive images at sub-pixel resolution (Fig. 1b; Hoechst is partially bleached by oxidation and we typically readd it at each cycle).

Bottom Line: Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers.Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited.Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.

View Article: PubMed Central - PubMed

Affiliation: HMS LINCS Center &Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 USA.

ABSTRACT
Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.

No MeSH data available.


Related in: MedlinePlus