Cd24 pacific blue

Manufactured by BioLegend

Sourced in United States

CD24-Pacific Blue is a fluorochrome-conjugated antibody that binds to the CD24 surface antigen. CD24 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein expressed on various cell types, including B cells, neutrophils, and some stem cells. The Pacific Blue fluorochrome is used to label the CD24 antibody, enabling flow cytometric detection and analysis of CD24-positive cells.

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Lab products found in correlation

Cd45 pe, by Thermo Fisher Scientific (4 mentions) Cd31 pe, by BioLegend (4 mentions) Ter119 pe, by BioLegend (4 mentions) Epcam apc, by Thermo Fisher Scientific (3 mentions) Ter119, by BD (3 mentions) 7 aad, by Thermo Fisher Scientific (2 mentions) Tryple, by Thermo Fisher Scientific (2 mentions) Cd117 apc cy7, by BioLegend (2 mentions) Facs 2 sorp cell sorter, by BD (2 mentions) Ab56416, by Merck Group (1 mentions) Chloroquine c6628, by Merck Group (1 mentions) Pm036, by Progen Biotechnik (1 mentions) Alexa fluor 647 conjugated goat anti mouse, by Merck Group (1 mentions) Tat beclin 1, by Selleck Chemicals (1 mentions) Alexa fluor 568 conjugated goat anti mouse, by Merck Group (1 mentions) Propidium iodide, by Merck Group (1 mentions) Hoechst 33342, by Merck Group (1 mentions) Igm pe cy5, by BD (1 mentions) Cd24 pe cy7, by BioLegend (1 mentions) Cd19 pacific blue, by BioLegend (1 mentions)

Spelling variants of this product

Pacific Blue anti-mouse CD24 (4 citations) Pacific-Blue-conjugated anti-CD24 (2 citations)

7 protocols using cd24 pacific blue

Isolation and Flow Cytometry of Intestinal Stem Cells

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Following crypt isolation, the crypt suspensions were pelleted (100 g, 5 min, 4°C) and supernatant was discarded. Crypts were resuspended in TrypLE (GIBCO, no phenol red, 12604039) and dissociated into individual cells by warming crypts in water bath at 32°C for 60 s. Dissociated single cells were treated with the following antibody cocktail for flow cytometry analysis: CD45-PE (eBioscience, 12-0451-83), CD31-PE (Biolegend, 102514), Ter-119PE (Biolegend, 116208), CD24-Pacific Blue (Biolegend, 101820), CD117-APC/Cy7 (Biolegend, 105826), and EpCAM-APC (eBioscience, 17-5791-82). 7AAD (Invitrogen, A1310) was used a viability dye to exclude dead cells from the analysis. ISCs were isolated as Lrg5-eGFP^hiEpCAM⁺CD24^low/−CD31⁻Ter119⁻CD45⁻7AAD⁻. eGFP^low progenitors were isolated as Lrg5-eGFP^lowEpCAM⁺CD24^low/−CD31⁻Ter119⁻CD45⁻7AAD⁻. Cells were sorted using a BD FACS II SORP cell sorter.

Mana M.D., Hussey A.M., Tzouanas C.N., Imada S., Millan Y.B., Bahceci D., Saiz D.R., Webb A.T., Lewis C.A., Carmeliet P., Mihaylova M.M., Shalek A.K, & Yilmaz Ö.H. (2021). High-fat diet-activated fatty acid oxidation mediates intestinal stemness and tumorigenicity. Cell reports, 35(10), 109212.

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Immunofluorescence and Western Blot Protocols

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Primary antibodies for western blot were against LC3 (Novus Biologicals, NB600), SQSTM1 (Abcam, ab56416), ATG13 (Sigma Aldrich, SAB4200100) and ACTIN (EMD Millipore, MAB1501). Primary antibodies used for immunofluorescence and immunocytochemistry were against LC3 (MBL, PM036), SQSTM1 (Progen, GP62-C), ATG16L1 (Abgent, AP1817b), AQP5 (Alomone labs, AQP5-005) and BrdU (Bio-Rad, MCA2483GA). Fluorescently-labeled antibodies for FACS sorting were against PECAM1/CD31 (PECAM1/CD31-PE; eBioscience, 12–0311-82), PTPRC/CD45 (PTPRC/CD45-PE, Biolegend, 103106), LY76/TER119 (LY76/TER119-PE/Cy7; Biolegend, 116222), CD24 (CD24-Pacific Blue, Biolegend, 101820) and ITGB1/CD29 (ITGB1/CD29-FITC, BD Biosciences, 555005). The following secondary antibodies from ThermoFisher Scientific were used for the visualization of the primary antibodies: Alexa Fluor 488-conjugated goat anti-mouse (A-11001), Alexa Fluor 568-conjugated goat anti-mouse (A-11031) or goat anti-rabbit (A-11011), Alexa Fluor 647-conjugated goat anti-mouse (A-21235), and Alexa Fluor 568 conjugated goat anti-Guinea pig (A-11075).
Hoechst 33342 was from Sigma Aldrich (B2261), Chloroquine (C6628) was from Sigma Aldrich and bafilomycin A₁ was from BioAustralis (BIA-B1012). Tat-beclin 1 was from Selleck Chemicals (S8595), while propidium iodide was from Sigma Aldrich (P4170).

Orhon I., Rocchi C., Villarejo-Zori B., Serrano Martinez P., Baanstra M., Brouwer U., Boya P., Coppes R, & Reggiori F. (2021). Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal. Autophagy, 18(2), 293-308.

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Flow Cytometric Immunophenotyping of Immune Cells

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For flow cytometry, cells were stained with mAbs in PBS with 1% BSA for 20 minutes in the dark at 4°C. Cells were subsequently washed and fixed with a 1% paraformaldehyde/PBS solution before analysis on BD FACSCalibur flow cytometer (BD Immunocytometry Systems, [BDIS] San Jose, CA) or MACSQuant® Analyzer (Miltenyi Biotec). The anti-human antibodies used were CD19 Pacific Blue, CD24 PE-Cy7, CD38 APC, CD27 APC-Cy7, CD21 FITC, CD1c PE (all from Biolegend, San Diego, CA), CD10 PE, CD5 PE, CD23 PE, IgD PE, IgM PE-Cy5 (all from BD Bioscience), BAFF-R PE (eBioscience Inc., San Diego, CA). The following anti-mouse antibodies were used: CD24 Pacific Blue, CD21 FITC, CD23 PE, BAFF-R PE, CD21 APC, SA-APC-Cy7 (secondary antibody for IgD biotin staining), IgM PE-Cy7 (all from Biolegend), and IgD Biotin (eBioscience). Living cells were identified by forward scatter and side scatter gating and/or exclusion of 7-aminoactinomycin-D (eBioscience) added immediately prior to data acquisition or fixation. Flow cytometry data analysis was performed using Flowjo data analysis software (TreeStar, Ashland, OR).

Benitez A., Weldon A.J., Tatosyan L., Velkuru V., Lee S., Milford T.A., Francis O.L., Hsu S., Nazeri K., Casiano C.M., Schneider R., Gonzalez J., Su R.J., Baez I., Colburn K., Moldovan I, & Payne K.J. (2014). Differences in Mouse and Human Non-Memory B Cell Pools. Journal of immunology (Baltimore, Md. : 1950), 192(10), 4610-4619.

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Isolation and Flow Cytometry of Intestinal Stem Cells

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Isolation of Splenic CD8α+ and pre-cDC1 Cells

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Splenic pan DCs were isolated from nine age-matched, female, littermate BALB/c mice and pooled per three mice for a total of three biological replicates. Pooled DCs were washed in flow buffer (PBS with 0.5% FBS), incubated with anti-mouse Fc Block (Thermo Fisher Scientific), and then stained with CD8α PE-Cy7 (Thermo Fisher; clone: 53–6.7), and CD24 Pacific Blue (Biolegend; clone: M1/69). CD8α+ and pre-cDC1 (CD24^high CD8α-) were sorted using FACSAria III (BD).

Molina M.S., Hoffman E.A., Stokes J., Kummet N., Simpson R.J, & Katsanis E. (2022). Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway. PLoS ONE, 17(8), e0273075.

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Isolation of Intestinal Stem Cells and Paneth Cells

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Intestinal stem cells and Paneth cell isolation was performed as described previously (Igarashi & Guarente, 2016; Roth et al., 2012: Yilmaz et al., 2012). Briefly, the crypt suspensions were centrifuged for 5 min at 250 g at 4°C. The pellets were gently resuspended in 1.0 ml of undiluted TrypLE Express (Life Technologies) +120 μl of DNase I (10 U/μl, Roche). The suspended crypts were incubated in a 32°C water bath for 1.5 min were then placed on ice. 12 ml of cold MEM was added, and crypts were gently triturated twice. After centrifugation, the pellets were resuspended and incubated for 15 min on ice in 1 ml MEM containing CD45‐PE (1/500 eBioscience), CD31‐PE (1/500 Biolegend), Ter119‐PE (1/500 Biolegend), and CD24‐Pacific Blue (1/500 Biolegend). After centrifugation, the pellets were resuspended with MEM containing 1.5 μM propidium iodide (PI) (Thermo Scientific). The samples were filtered through a 40‐μm mesh (Corning) and immediately sorted on a FACS Aria (Becton Dickinson).
Intestinal stem cells were isolated as Lgr5‐EGFP^hiCD24^low/−CD31⁻Ter119⁻CD45⁻PI⁻,and Paneth cells were isolated as CD24^hiSideScatter^hiLgR5‐EGFP⁻CD31⁻Ter119⁻CD45⁻PI⁻ (Igarashi & Guarente, 2016).

Igarashi M., Miura M., Williams E., Jaksch F., Kadowaki T., Yamauchi T, & Guarente L. (2019). NAD+ supplementation rejuvenates aged gut adult stem cells. Aging Cell, 18(3), e12935.

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Intestinal Epithelial Cell Isolation

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After 6 h of recovery from surger, alive mice were sacrificed via cervical dislocation, and PCs were harvested from crypts of the ileum, as previously described [18] . In brief, crypts from the ileum were mainly collected from mixed intestinal fragments through a 70-μm mesh filter. Next, the crypt suspensions were marked with an antibody cocktail consisting of CD45-PE, EPCAM-APC (eBioscience, CA, USA), CD31-PE, Ter119-PE, CD31-PE, Ter119-PE and CD24-Pacific Blue (BioLegend, CA, USA), and resuspended with SEME/7-aminoactinomycin D (7-AAD) solution (1:500 dilution), followed by flow cytometry selection and analysis using a forward and side scatter gating strategy (BD Biosciences, San Jose, CA, USA). PCs were isolated as CD24 hi Epcam + CD31 -Ter119 -CD45 -7-AAD - cells, with the percentage in the crypt suspensions recorded. Dead cells were abandoned for subsequent studies, using the viability dye 7-AAD.

Zhang J., Ye J., Ren Y., Zuo J., Dai W., He Y., Tan M., Song W, & Yuan Y. (2018). Intracellular activation of complement C3 in Paneth cells improves repair of intestinal epithelia during acute injury. Immunotherapy, 10(15).

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