MA5-13890

antibody from Invitrogen Antibodies

Targeting: CD44 CD44R, CSPG8, HCELL, IN, MC56, MDU2, MDU3, MIC4, Pgp1

Provider product page for MA5-13890

Western blot

Immunoprecipitation

Antibody data

Antibody Data
Antigen structure
References [77]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [5]
Immunohistochemistry [2]
Flow cytometry [2]
Other assay [18]

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Product number: MA5-13890 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD44 Monoclonal Antibody (156-3C11)
Antibody type: Monoclonal
Antigen: Other
Description: MA5-13890 targets CD44 in FACS, IHC (P), ICC/IF, IP and WB applications and shows reactivity with Human, Baboon and Non-human primate samples. The MA5-13890 immunogen is stimulated human leukocytes. MA5-13890 was used in the immunofluorescence analysis to successfully detect CD44 in human mesenchymal stem cells derived from iPSCs.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 156-3C11
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

CD44 protein structure - MA5-13890 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of mesenchymal stem cell marker CD44 (green) in human mesenchymal stem cells CY-2 derived from iPSCs. The cells were fixed with 4% Paraformaldehyde for 30 mins and blocked with blocking buffer for 1 hour at room temperature. Cells were stained with CD44 mouse monoclonal antibody (Product # MA5-13890) at a dilution of 1:100 in staining buffer overnight at 4°C, and then incubated with secondary antibody AlexaFluor 488 donkey anti-mouse (Product # A-21202) at a dilution of 1:250 for 1 hour at room temperature. Nuclear (blue) were stained with NucBlue Fixed Cell Stain (Product # R37606). Images were taken at 20X magnification. Data courtesy of Dr. Wei Zheng's lab.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of CD44 Std (green) showing staining in the membrane of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD44 Std monoclonal antibody (Product # MA5-13890) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of CD44 Std (green) showing staining in the membrane of MDA-MB-231 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD44 Std monoclonal antibody (Product # MA5-13890) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of CD44 Monoclonal Antibody was performed using 70% confluent log phase Raji cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with CD44 (156-3C11) Mouse Monoclonal Antibody (Product # MA5-13890) at 2 µg/mL in 0.1% BSA, incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membranous localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence of CD44 was performed using HAP1 wild-type and CD44 KO cells that were transfected with a green or a far red fluorescent dye, respectively. Post-transfection, WT and KO cells were mixed and plated to a 1:1 ratio on coverslips as a mosaic and incubated for 24 hrs. Cells were fixed in 4% PFA (in PBS) for 15 min; cells were permeabilized with 0.1% Triton X-100 for 10 min at RT and blocked with PBS with 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 min. Cells were stained with the CD44 monoclonal antibody (Product # MA5-13890) at a 1:200 dilution overnight at 4°C. Secondary antibody incubation was performed using 0.5 µg/mL of Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 antibody (Product # A21424) together with DAPI for 1 hr. Imaging was performed with a 40X oil objective and analysis was performed using Image J. Cell image represents a single focal plane; WT and KO cells are outlined with a yellow (WT) or magenta (KO) dashed line. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of CD44 Std using CD44 Std Monoclonal Antibody (Product # MA5-13890) on Native Human HeLa Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 PCa cell lines profiled for REST expression and characterization of EMT and stemness signature. ( A and B ) LNCaP, CWR22Rv1, PC3, and DU145 were examined for REST protein by immunoblotting ( A ) and REST mRNA by RT-qPCR ( B ). n = 3. ( C ) Immunoblotting of protein expression for REST, NE marker SYP, and EMT and stemness markers as indicated in LNCaP and CWR22Rv1 cells. n = 3. ( D ) mRNA levels of NE and EMT markers and CD44 in LNCaP and CWR22Rv1 cells. n = 2. ( E and F ) Immunofluorescence micrographs ( E ) and flow cytometric analysis ( F ) of CD44 expression in LNCaP and CWR22Rv1 cells. n = 2. ( G and H ) LNCaP and CWR22Rv1 cells were seeded in transwell (1 x 10 4 ) or cultured in defined serum-free sphere medium with growth factors (2 x 10 4 ) and measured at 3 and 14 days, respectively. The number of migrated cells was quantified by the average from 5 microscopic fields ( G ). The number of spheres was calculated ( H ). Representative images (left) and quantification (right) in both PCa cell lines. n = 3. Data represent means +- SD. ** p < 0.01, *** p < 0.001 by Student's-t test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Short-term overexpression of REST inhibits EMT and stemness properties of CWR22Rv1 cells. ( A ) Immunoblotting of REST, NE marker SYP, and EMT and stemness markers in CWR22Rv1-TR-REST cells receiving 1 mug/ml Dox for 3 days (P1) compared to no Dox control. n = 3. ( B ) CWR22Rv1-TR-REST cells treated as described in ( A ) were seeded on the transwell membrane and after three days the cells were stained with Hoechst. Representative images (left) and quantification (right) of migrated CWR22Rv1 cells. n = 3. ( C and D ) Immunofluorescent micrographs ( C ) and flow cytometric analysis ( D ) of CD44 expression in CWR22Rv1 cells treated as described in ( A ). n = 2. ( E ) CWR22Rv1-TR-REST cells treated as described in ( A ) were then cultured in defined serum-free sphere medium for 14 days. Representative images (left) and quantification (right) of sphere formation in CWR22Rv1 cells. n = 3. Data represent means +- SD. *** p < 0.001 by Student's-t test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Long-term overexpression of REST inhibits EMT and stemness properties of CWR22Rv1 cells. ( A ) Immunoblotting of REST, NE marker SYP, and EMT and stemness markers in CWR22Rv1-TR-REST cells receiving 1 mug/ml Dox for 45 days (P15) compared to no Dox control. n = 2. ( B ) CWR22Rv1-TR-REST cells treated as described in ( A ) were seeded on the transwell membrane and after three days the cells were stained with Hoechst. Representative images (left) and quantification (right) of migrated CWR22Rv1 cells. n = 3. ( C and D ) Immunofluorescent micrographs ( C ) and flow cytometric analysis ( D ) of CD44 expression in CWR22Rv1 cells treated as described in ( A ). n = 2. ( E ) CWR22Rv1-TR-REST cells treated as described in ( A ) were then cultured in defined serum-free sphere medium for 14 days. Representative images (left) and quantification (right) of sphere formation in CWR22Rv1 cells. n = 3. Data represent means +- SD. *** p < 0.001 by Student's-t test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of CD44 was performed on HAP1 cell lysates. Antibody-bead conjugates were prepared by adding 1 µg of CD44 recombinant monoclonal antibody (Product # MA5-13890) with 30 µL of protein G-Sepharose beads and rocked overnight at 4°C. 1 mg of lysate was incubated with an antibody-bead conjugate for 2 hours at 4°C. Following centrifugation and multiple washes, 10% starting material (SM), 10% unbound fraction (UB) and immunoprecipitated fraction (IP) were processed for immunoblot using a different CD44 monoclonal antibody. Ponceau stained transfer of blot is shown. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 CD9 + PMPs on BCCs A. Confocal images were taken every 25 min of live CD9-GFP-transfected MDA cells cultured with DiD-labeled MSCs. Retraction of magnupodium (asterisk) occurs on a 1 h-time scale. Arrows, branched CD9 + filopodia detached from cells and degraded over time. B . C . Fixed, permeabilized MDA cells were stained with anti-CD9 Ab followed by Cy5-conjugated secondary Ab. Images are maximum intensity projections (MIPs) of confocal z-stacks. Representative z-optical sections taken at different heights from the coverslip shown to the right of the MIP. Arrow, branched CD9 + filopodia; white asterisk, lamellipodium at the tip of magnupodium (B). CD9 + PMPs such as a lamellipodium (arrow) at the tip of magnupodium and short filopodia (arrowheads) promote the intercellular interaction between two MDA cells. High power views of images of outlined areas are shown (C). D . CD9-GFP-transfected MDA cells stained with anti-CD44 Ab followed by Cy5-conjugated secondary Ab. Image is representative z-optical section of microvilli. Insets show GFP and Cy5 channels. E - G . CD9 + PMPs on MDA-CD9-GFP, MA-11-CD9-GFP, and MCF-7-CD9-GFP cells. Branched CD9 + filopodia (arrowhead) are emerging from MDA cells over long distance (see magnification of the outlined area) (E). Short CD9 + filopodia (arrowhead) are emerging from MA-11 (F) and MCF-7 (G). Note numerous CD9 + long microvilli in MA-11 cells and large dorsal ruffles (intense green areas) in MCF-7 cells (F, G, respectively, s

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Cell cycle is accelerated and the apoptosis rate is suppressed by CHIR and PD in HMLE cells. (A) Cell cycle distribution and apoptosis rate in HMLE cells following treatment with CHIR and PD were determined using flow cytometry analysis. (B) Flow cytometry analysis of CD44 and CD24 for control cells, and cells treated with CHIR and PD. All assays were performed in duplicates and data are presented as the mean +- standard deviation. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Immunohistochemical double-staining of human breast tumors for CD44 and CD24. CD44 is stained with Permanent Red and CD24 with diaminobenzidene (DAB). Magnification x 20. (a) A tumor positive for both CD44 + /CD24 - (white arrow) and CD44 - /CD24 + (black arrow) cancer cells, although the predominant phenotype is CD44 - /CD24 - . (b) Almost all cells in this tumor are CD44 + /CD24 - . No CD24 staining is seen. (c) A tumor with predominantly CD44 + /CD24 - cells. A few CD44 + /CD24 + cells are also present (black arrow). (d) A tumor positive for the CD44 - /CD24 + phenotype. No CD44 staining is present.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 The in vivo invasion analysis using the chicken chorioallantoic membrane (CAM) assay. ( a - f ) CD44 stained OV-90 cells transfected with ( a ) empty vector (pcDNA3.1(+)) and overexpression constructs, ( b ) PIWIL1 , ( c ) P117 , ( d ) PIWIL2 , ( e ) PL2L60 and ( f ) MAEL . CAMs were counterstained with haematoxylin. Invasion was quantified by the number of OV-90 cells that successfully breached the ECT and beyond (* marked next to breached area). CM = cancer cells mixed with matrigel, ECT = ectoderm, MES = mesoderm, END = endoderm. Scale bar = 100 um. ( g ) Significantly more CAM area was invaded by P117 and PL2L60 overexpressing OV-90 cells. Both mutants were more invasive than its respective wildtype form where only overexpression of P117 was significant. n = 4-18 per construct transfected and carried out in biological duplicates. Statistical tests Mann-Whitney U (non-normal distribution) or unpaired t -test (normal distribution) were used accordingly. * p < 0.05; *** p < 0.0005.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 8 TRIM59 nuclear translocation is required for GSC tumorigenicity. a Effects of EGFR inhibitor erlotinib or CDK5 inhibitor Roscovitine treatment on p-EGFR, p-CDK5, p-STAT3, and mH2A1 protein levels in GSC R39 and R83 cells. Arrows, EGFR. Arrow heads, EGFRvIII. b qRT-PCR analyses of erlotinib or Roscovitine treatment on STAT3-targeted gene PIM1 mRNA expression. c Limiting dilution glioma sphere-forming assays in GSC R39 and R83 cells with erlotinib or Roscovitine treatment. d - f Effects of TRIM59 shRNA knockdown and re-expression of shRNA-resistant TRIM59 WT, S308A, or S308D mutant on TRIM59, mH2A1 protein levels ( d ), p-STAT3 ( d ), STAT3-targeted gene PIM1 expression ( e ), and glioma sphere formation ( f ) in GSC R39 and R83 cells. g , h Representative bioluminescence images of tumor growth ( g ) and quantification of bioluminescence ( h ). n = 5. i Representative images of IHC analysis of indicated protein expression in GSC R39 xenograft tumors. Scale bar, 50 mum. j , k Effects of TRIM59 shRNA knockdown and re-expression of shRNA-resistant TRIM59 WT, S308A, or S308D mutant on TRIM59, mH2A1 protein levels ( j ), p-STAT3 ( j ), and STAT3-targeted gene PIM1 expression ( k ) in GSC R39 xenograft tumors. Data are representative of three independent experiments with similar results. Data were expressed as means +- SD. * P < 0.05, ** P < 0.01, by two-tailed Student's t test. Source data are provided as a Source Data file

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Endogenous stem cells recruitment by HCLS. a1 Schematic depiction of in vitro BMSCs recruitment by incubating various scaffolds in a total rabbit cranial bone marrow for 48 h. a2 CD44 immunofluorescent staining imaged by CLSM. a3 Schematic depiction of transwell assay in studying BMSCs migration to various scaffolds. a4 Quantification of migrated BMSCs by dissolving crystal violet and spectrophotometrically measured at 573 nm, the optical density (OD) resulted was normalized by control. BMSCs alone was used as control (** p = 0.0028, *** p = 0.0001, *** p = 0.0002, *** p = 6.7070 x 10 -5 ). n = 3 biologically independent replicates. b SEM images of endogenous cells on the surface of various scaffolds. c CLSM images (rhodamine-phalloidin/DAPI staining) of endogenous cells on the surface of various scaffolds. d CLSM images of CD44/F-actin/DAPI staining of various scaffolds. e1 Confocal quantitative images (CD44/DAPI staining) of ESCs in scaffolds. e2 High-magnification images (CD44/DAPI staining) of ESCs in scaffolds. e3 Quantitative analysis of the total number of cells in scaffolds (* p = 0.0470, ** p = 0.0023). e4 Quantitative analysis of the number of ESCs in scaffolds (*** p = 0.0008, *** p = 0.0003). n = 3 biologically independent replicates. f1 - 3 BMP-2 and Runx2 immunofluorescence staining at one week of different treatments and semiquantitative analysis ( f1 : *** p = 0.0009, *** p = 5.5298 x 10 -5 ; f2 : ** p = 0.0078, *** p = 5.8847 x 10 -5 ). g1 CLSM images of CD31

Supportive validation

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Experimental details: Figure 3 Immunohistochemistry in breast tumor sections from triple-negative breast cancer (TNBC) patients for LSD1 and the stemness marker CD44. ( A - D ) Representative images from four different tumors (cases 4, 8, 9, and 10 in Table S1 ) showing high enrichment in CD44 + cells and high co-expression of CD44 and LSD1. Magnification 200x. ( E ) Very high (-100%) and ( F ) lower (-40%) concomitant expression of LSD1 and CD44 in neoplastic cells. ( G ) Strong and ( H ) moderate nuclear staining for LSD1 in neoplastic cells. Magnification 600x. ( I , J ) Non-neoplastic cells of the adjacent breast parenchyma with weak nuclear LSD1 staining from two different tumors (magnification 400x). The cell surface antigen CD44 exhibits membranous staining (chromogenic substrate: magenta) and LSD1 is nuclear (chromogenic substrate: DAB).

Supportive validation

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Experimental details: Figure 5 LSD1 inhibition reduced tumor growth and targeted the CSC subpopulation in vivo. MDA-MB 468 breast cancer cells were injected orthotopically in mice (into two fat pads/mouse). Treatment with the GSK-LSD1 inhibitor ( n = 6) or with the vehicle control ( n = 5) started when the tumors were detectable by palpation and lasted for 22 days, when the tumors were removed and measured. ( A ) Representative images of the tumors excised from mice treated with vehicle or GSK-LSD1 inhibitor. ( B ) Graphical representation of changes in tumor size over the course of the experiment. ( C ) Graphical representation of the effects LSD1 inhibition on the mean tumor volume as measured at the end of the experiment. ( D ) The FACS analysis of dissociated tumor cells isolated pre-treatment or post-treatment with GSK-LSD1 or vehicle. Single cells were stained with antibodies against the CD44 and CD24 cell surface proteins. ( E ) Quantification of FACS analysis presented in ( D ). Error bars represent SEM. *** p < 0.001, **** p < 0.0001.