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Calcein blue

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Calcein blue is a fluorescent dye used in various laboratory applications. It is a calcium-sensitive indicator that emits blue fluorescence upon binding to calcium ions. The core function of calcein blue is to facilitate the detection and quantification of calcium levels in biological samples.

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Lab products found in correlation

Propidium iodide, by Thermo Fisher Scientific (2 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) Trustain fcx, by BioLegend (2 mentions) Anti human gpi 80 pe antibody, by MBL Life Science (2 mentions) Anti human cd235a apc antibody, by BD (2 mentions) Anti human cd34 apc antibody, by BD (2 mentions) Facsaria 3, by BD (2 mentions) Diva software, by BD (2 mentions) Collagenase, by Thermo Fisher Scientific (2 mentions) Dispase, by Thermo Fisher Scientific (2 mentions) Anti mouse cd11b pe conjugate, by Thermo Fisher Scientific (2 mentions) Anti mouse sca 1 pe conjugate, by BioLegend (2 mentions) Anti mouse cd31 pe cy7 conjugate, by BioLegend (2 mentions) Collagenase, by Harvard Bioscience (2 mentions) Anti cd90.2 pe cy7, by Thermo Fisher Scientific (2 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Fc block, by BD (1 mentions) Tryple, by Thermo Fisher Scientific (1 mentions) Stain buffer, by BD (1 mentions) Ultracomp ebeads compensation beads, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Calcein (176 citations) Calcein Blue AM (13 citations)

17 protocols using calcein blue

1

Mammary Gland Flow Cytometry

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For flow cytometry experiments, mammary glands were excised and digested as described for organoid experiments. Following digestion at 37 °C, samples were dissociated into single cells using TrypLE (Gibco, 12604-021), quenched with DMEM/10% FBS, and washed with PBS. Cells were then put through a 40 µM (Fisher, 22-363-547) or 70 µM strainer (Corning, 352350). For mammary gland profiling, live/dead staining was performed using a near-IR dead cell stain (Invitrogen, L34976), followed by blocking using Fc block (BD Biosciences, 553142) in BD stain buffer (BD, 554656). Samples were then incubated in primary antibodies at the specified concentrations for 30 min at 4 °C, washed twice, and resuspended in PBS. Compensation was performed using UltraComp eBeads Compensation Beads (Invitrogen). Flow cytometry was performed using a BD LSRII flow cytometer using FACS Diva 6.2.1 (BD Biosciences) and analysis was performed using FlowJo v10.7.1 (BD Biosciences). All antibodies used for flow cytometry are outlined in Supplementary Table 2. For FACS experiments, live/dead staining was performed using Calcein blue (Invitrogen, C1429) at a concentration of 1 µM. Cell sorting was performed on a Moflo Astrios flow cytometer (Beckman Coulter).
Kern J.G., Tilston-Lunel A.M., Federico A., Ning B., Mueller A., Peppler G.B., Stampouloglou E., Cheng N., Johnson R.L., Lenburg M.E., Beane J.E., Monti S, & Varelas X. (2022). Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas. Nature Communications, 13, 7198.
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2

Isolation of Muscle and Tumor Cell Subsets

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Phenotypically distinct muscle and tumor cell subsets were sorted according to protocols that were previously established to isolate functionally discrete subsets of myofiber-associated cells [10 (link), 11 (link), 1 (link)]. In brief, cells were suspended in HBSS supplemented with 2% FBS. Antibody staining was performed for 20 minutes on ice. The following primary and secondary antibodies were used: APC-CY7-conjugated anti-mouse CD11b (1 in 200, BD Pharmingen, 557657), APC-CY7-conjugated anti-mouse CD45 (1 in 200, eBioscience, 557659), APC-CY7-conjugated anti-mouse TER119 (1 in 200, BioLegend, 116223), APC-conjugated anti-mouse Sca1 (1 in 200, eBioscience, 17-5981-82), PE-conjugated anti-mouse/ rat CD29 (1 in 400, BioLegend, 102207), biotin rat anti-mouse CD184 (1 in 100, BD Pharmingen, 551968), PECY7-conjugated Streptavidin (1 in 200, eBioscience, 25-4317-82). Antibody staining was performed for 20 minutes on ice. Prior to FACS sorting, cells were suspended in 1µg/ml propidium iodide and 10µM calcein blue (Invitrogen) to identify viable cells (Pi−Ca+). Cells were sorted twice to maximize purity.
Hettmer S., Lin M.M., Tchessalova D., Tortorici S.J., Castiglioni A., Desai T., Mao J., McMahon A.P, & Wagers A.J. (2015). Hedgehog-driven myogenic tumors recapitulate skeletal muscle cellular heterogeneity. Experimental cell research, 340(1), 43-52.
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3

Isolation of GFP+ and GFP- Cells in Zebrafish

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GFP+ and GFP− cells were isolated from pdgfrb:GFP transgenic zebrafish at 4 dpf. 120 animals were pooled for each experiment. Before tissue dissociation, head, yolk sac, and tail fin were removed, using micro scissors. Dissociation, live cell staining, and FACS sorting of cells were done as previously described13 (link). Briefly, excised tissue was enzymatically dissociated using 0.25% Trypsin-EDTA (Gibco Cat#25200072) followed by mechanical dissociation using a fire-polished glass Pasteur pipette. Cell suspension was filtered through a 20 µm cell strainer (pluriSelect Cat#43-10020-40). Following centrifugation for 10 min at 300 g, cells were resuspended in 500 µL HBSS medium (Gibco Cat#14065-056). To stain for viable cells, Calcein Blue (Invitrogen Cat#C1429) was added to the cell suspension. Cells were directly sorted into lysis buffer using a MoFlo Astrios EQ sorter (Beckman Coulter). For the detection of GFP, a 488 nm excitation laser and a 526/52 nm bandpass filter were used. Calcein Blue was detected following 405 nm excitation using a 431/28 nm bandpass emission filter.
Kolb J., Tsata V., John N., Kim K., Möckel C., Rosso G., Kurbel V., Parmar A., Sharma G., Karandasheva K., Abuhattum S., Lyraki O., Beck T., Müller P., Schlüßler R., Frischknecht R., Wehner A., Krombholz N., Steigenberger B., Beis D., Takeoka A., Blümcke I., Möllmert S., Singh K., Guck J., Kobow K, & Wehner D. (2023). Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment. Nature Communications, 14, 6814.
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4

Characterizing Muscle Stem Cells Post-Injury

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Injured and uninjured TA muscles from C57BL/6 mice were harvested at day 3 and 5 after CTX injection. Muscles were weighed and mononuclear cells were obtained by enzymatic digestion with 0.2% dispase and 0.05% collagenase II in DMEM (Invitrogen) at 37°C for 15 min. The cells were counted and the antibody staining was performed 30 min on ice in HBSS (Invitrogen) 2% DBS using appropriate combinations of the antibodies. APC Cy7: CD45 (BD, clone 30-F11, 1:200), CD11b (BD, clone M1/70, 1:200), TER119 (Biolegend, clone TER-119, 1:200). CXCR4 biotinilated (BD, clone 2B11/CXCR4, 1:100) followed by PE-Cy7 streptavidin (eBioscience, 1:200). APC conjugated Sca-1 (eBioscience, clone D7, 1:200). PE conjugated β1 integrin (BD, clone M1/69, 1:200) or purified β1 integrin (BD, clone M1/69, 1:100) followed by FITC conjugated goat anti-hamster IgG (eBioscience, 1:200) when PE conjugated CD210 (IL10RA, Biolegend, clone 1B1.3a, 1:20) antibody was used. Calcein Blue (Invitrogen) and PI were used to distinguish live cells.
Castiglioni A., Corna G., Rigamonti E., Basso V., Vezzoli M., Monno A., Almada A.E., Mondino A., Wagers A.J., Manfredi A.A, & Rovere-Querini P. (2015). FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration. PLoS ONE, 10(6), e0128094.
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5

Apoptosis Assessment with Annexin V

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Trapped cells were exposed to Annexin V Alexa Fluor 488 (Invitrogen) at room temperature in the dark room for 30 minutes followed by exchanging the reagents into mixed reagents of Annexin binding buffer (Invitrogen), Propidium Iodide (Invitrogen) and calcein blue (Invitrogen) for 10 minutes. The whole process was conducted at a flow rate of 3 μL min-1.
Kobayashi M., Kim S.H., Nakamura H., Kaneda S, & Fujii T. (2015). Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device. PLoS ONE, 10(11), e0139980.
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6

Single-Cell Profiling of Hematopoietic Cells

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FL cells were thawed and allowed to recover at 37 °C for an hour prior to staining. Cells were blocked with TruStain FcX (BioLegend, 422301) and stained with TotalSeq A antibody mix containing 1ug of each TotalSeq A antibody per condition (BioLegend, see Table S1 for a list of antibodies). Anti-human CD235a-APC antibody (BD Biosciences, 551336) was added to the CD34− fraction. The CD34+ fraction was stained with anti-human CD34-APC antibody (BD Biosciences, 555824) and anti-human GPI-80-PE antibody (MBL International, D087-5). All samples were stained with calcein blue (Invitrogen, C34853) for live/dead exclusion and cell populations were sorted (Beckman Coulter MoFlo Astrios) as shown in Fig. S1A prior to loading onto the 10x Genomics platform. Chromium Single-Cell 3ʹ Reagent Kit v3 with Feature Barcoding technology for Cell-Surface Protein was used and the recommendations from the manufacturer were followed as specified in 10x Genomics user guide document number CG000185, Rev B.
Vanuytsel K., Villacorta-Martin C., Lindstrom-Vautrin J., Wang Z., Garcia-Beltran W.F., Vrbanac V., Parsons D., Lam E.C., Matte T.M., Dowrey T.W., Kumar S.S., Li M., Wang F., Yeung A.K., Mostoslavsky G., Dries R., Campbell J.D., Belkina A.C., Balazs A.B, & Murphy G.J. (2022). Multi-modal profiling of human fetal liver hematopoietic stem cells reveals the molecular signature of engraftment. Nature Communications, 13, 1103.
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7

Isolation of Muscle Stem Cells

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Muscles from hindlimbs from young adult or aged mice were dissected and collected in PBS on ice. Muscles were rinsed with PBS, minced with scissors and incubated in DMEM with Collagenase (0.2%, Biochrom) for 90 min at 37°C and 70 rpm. Digested muscles were washed with PBS/10% FBS, triturated and incubated in Collagenase (0.0125%) and Dispase (0.4%, Life Technologies) for 30 min at 37°C and 100 rpm. The muscle slurry was diluted with PBS/10% FBS, filtered through 100 μm cell strainers and spun down at 500 g for 5 min. Cell pellets were resuspended in FACS buffer (HBSS/2% FBS) and filtered through 40 μm cell strainers and pelleted at 500 g for 5 min. Pellets were resuspended in FACS buffer and stained with anti-mouse CD45 PE conjugate (30-F11, eBioscience), anti-mouse CD11b PE conjugate (M1/70, eBioscience), anti-mouse Sca-1 PE conjugate (D7, BioLegend), anti-mouse CD31 PE/Cy7 conjugate (390, BioLegend) and anti-mouse α7-Integrin Alexa Fluor 647 conjugate (R2F2, AbLab) for 20 min at 4°C on a rotating wheel. Cells were washed with FACS buffer. Live cells were identified as calcein blue positive (1:1,000, Invitrogen) and propidium iodide negative (PI, 1 μg/ml, BD Biosciences). SCs were identified as CD45−Sca-1−CD11b−CD31−α7-Integrin+. Cell sorting was performed on a FACSAriaIII with Diva Software (BD).
Schwoerer S., Becker F., Feller C., Baig A.H., Koeber U., Henze H., Kraus J.M., Xin B., Lechel A., Lipka D.B., Varghese C.S., Schmidt M., Rohs R., Aebersold R., Medina K.L., Kestler H.A., Neri F., von Maltzahn J., Tuempel S, & Rudolph K.L. (2016). Epigenetic stress responses induce muscle stem cell aging by Hoxa9 developmental signals. Nature, 540(7633), 428-432.
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8

Isolation of Muscle Stem Cells

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Muscles from hindlimbs from young adult or aged mice were dissected and collected in PBS on ice. Muscles were rinsed with PBS, minced with scissors and incubated in DMEM with Collagenase (0.2%, Biochrom) for 90 min at 37°C and 70 rpm. Digested muscles were washed with PBS/10% FBS, triturated and incubated in Collagenase (0.0125%) and Dispase (0.4%, Life Technologies) for 30 min at 37°C and 100 rpm. The muscle slurry was diluted with PBS/10% FBS, filtered through 100 μm cell strainers and spun down at 500 g for 5 min. Cell pellets were resuspended in FACS buffer (HBSS/2% FBS) and filtered through 40 μm cell strainers and pelleted at 500 g for 5 min. Pellets were resuspended in FACS buffer and stained with anti-mouse CD45 PE conjugate (30-F11, eBioscience), anti-mouse CD11b PE conjugate (M1/70, eBioscience), anti-mouse Sca-1 PE conjugate (D7, BioLegend), anti-mouse CD31 PE/Cy7 conjugate (390, BioLegend) and anti-mouse α7-Integrin Alexa Fluor 647 conjugate (R2F2, AbLab) for 20 min at 4°C on a rotating wheel. Cells were washed with FACS buffer. Live cells were identified as calcein blue positive (1:1,000, Invitrogen) and propidium iodide negative (PI, 1 μg/ml, BD Biosciences). SCs were identified as CD45−Sca-1−CD11b−CD31−α7-Integrin+. Cell sorting was performed on a FACSAriaIII with Diva Software (BD).
Schwoerer S., Becker F., Feller C., Baig A.H., Koeber U., Henze H., Kraus J.M., Xin B., Lechel A., Lipka D.B., Varghese C.S., Schmidt M., Rohs R., Aebersold R., Medina K.L., Kestler H.A., Neri F., von Maltzahn J., Tuempel S, & Rudolph K.L. (2016). Epigenetic stress responses induce muscle stem cell aging by Hoxa9 developmental signals. Nature, 540(7633), 428-432.
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9

Intestinal Epithelial Cell Isolation

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To acquire the intestinal epithelial population, approximately 100 of 6dpf TgBAC(cldn15la:GFP) zebrafish larvae were collected for the FACS experiment, then intestines were dissected and placed into PBS on ice with a maximum dissection time of 2 h. Intestine dissociation was performed using TrypLE Express (Gibco, Cat. No. 12605028) for 1 h at 37 °C, pipetting up and down every 10 min to support digestion. Digested samples were spun at 1500 g for 5 min at 4 °C and washed twice with PBS 1× before being resuspended together with PBS 1× and 10% FBS (fetal bovine serum). Filtered cells were immediately subjected to FACS at Institut Curie Flow Cytometry Platform using the Sony SH800 Cell Sorter. Dead cells were excluded from analysis using the combination of Calcein Blue (Invitrogen, Cat. No. 65-0855-39) and Propidium Iodide viability stains (Sigma-Aldrich, Cat. No. P4864). Non-transgenic and single transgenic controls (pools of 10 dissected guts) were prepared as above and used for gating and compensation. RNA isolation was done using on average 30,000 GFP+ or GFP− sorted cells with the Single-Cell RNA Purification Kit from Norgen Biotek Corp and reverse transcribed using Superscript IV Reverse Transcriptase (Life Technologies, Cat. No. 18090050) following the manufacturer’s instructions.
Morales R.A., Rabahi S., Diaz O.E., Salloum Y., Kern B.C., Westling M., Luo X., Parigi S.M., Monasterio G., Das S., Hernández P.P, & Villablanca E.J. (2022). Interleukin-10 regulates goblet cell numbers through Notch signaling in the developing zebrafish intestine. Mucosal Immunology, 15(5), 940-951.
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10

Retina Single-Cell Isolation and FACS

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Retinas were dissected in AMES solution (Sigma A1420, equilibrated with 95% O2/5% CO2). Upon dissection, eyes and lenses were visually inspected for damage, blood, or inflammation, which were used as criteria for exclusion. Retinas were digested in papain and dissociated to single cell suspensions using manual trituration in ovomucoid solution. Cells were spun down at 450 g for eight minutes, resuspended in AMES+4% BSA to a concentration of 10 million cells per 100ml. 0.5ml of 0.2mg/ml anti-CD90.2-PE-Cy7 (Affymetrix eBioscience 25-0902-82) per 100ml of cells was incubated for 15 min, washed with an excess of media, spun down and resuspended again in AMES+4% BSA at a concentration of ~7 million cells per 1 ml. Just prior to FACS the live cell marker Calcein Blue (Invitrogen C1429) was added. Cellular debris, doublets, and dead cells (Calcein Blue negative) were excluded. For FACS experiments with vGLUT2-Cre; LSL-Cas9 mice, RGCs were collected based CD90.2 and GFP double positive expression. For purification of transduced RGCs with CRISPR perturbation (sgRNAs co-expressed with mcherry), cells were collected based on CD90.2, GFP and mcherry triple positive expression. Cells were collected into ~150ul of AMES+5% BSA.
Tian F., Cheng Y., Zhou S., Wang Q., Monavarfeshani A., Gao K., Jiang W., Kawaguchi R., Wang Q., Tang M., Donahue R., Meng H., Zhang Y., Jacobi A., Yan W., Yin J., Cai X., Yang Z., Hegarty S., Stanicka J., Dmitriev P., Taub D., Zhu J., Woolf C.J., Sanes J.R., Geschwind D.H, & He Z. (2022). Core Transcription Programs Controlling Injury-Induced Neurodegeneration of Retinal Ganglion Cells. Neuron, 110(16), 2607-2624.e8.
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