Anti-CD14 antibody [EPR21847]

• Flow Cyt

Lab

Flow Cytometry - Anti-CD14 antibody [EPR21847] (AB221678)

Flow cytometry staining of C57 BL/6 mouse bone marrow cells with ab221678 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Cells were incubated for 30 min on ice in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab221678 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 106 in 100 μl at 10.0 μg/ml (1/215)) for 30min on ice. The cells were simultaneously stained with Ly6G.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.

• Flow Cyt

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Flow Cytometry - Anti-CD14 antibody [EPR21847] (AB221678)

Flow cytometric analysis of RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cell line labeling CD14 with ab221678 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells incubated with secondary antibody only) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488)(ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD14 antibody [EPR21847] (AB221678)

Immunofluorescent analysis of 100% methanol-fixed J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) cells labeling CD14 with ab221678 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining on J774A.1 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD14 antibody [EPR21847] (AB221678)

Immunofluorescent analysis of 100% methanol-fixed RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling CD14 with ab221678 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488)(ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining on RAW 264.7 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (re

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488)(ab150077) secondary at 1/1000 dilution.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-CD14 antibody [EPR21847] (AB221678)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen tissue labeling CD14 with ab221678 at 1/50 (10.62 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) at 1/1000 dilution (Green). Positive staining on mouse spleen. is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor ®; 488) at 1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IP

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Immunoprecipitation - Anti-CD14 antibody [EPR21847] (AB221678)

CD14 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab221678 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab221678 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10 μg (Input).
Lane 2 : ab221678 IP in RAW 264.7 whole cell lysate(+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab221678 in RAW 264.7 whole cell lysate (-).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 8 seconds

The molecular mass observed is consistent with the literature (PMID : 9502426; PMID : 7513013)

All lanes:

Immunoprecipitation - Anti-CD14 antibody [EPR21847] (ab221678)

Predicted band size: 40 kDa

Observed band size: 50-55 kDa

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• WB

Supplier Data

Western blot - Anti-CD14 antibody [EPR21847] (AB221678)

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : Lane 1 : 6 seconds; Lane 2 : 3 minutes; Lane 3 : 10 seconds; Lane 4 : 81 seconds.

The molecular mass observed is consistent with the literature (PMID : 9502426; PMID : 7513013).

All lanes:

Western blot - Anti-CD14 antibody [EPR21847] (ab221678) at 1/1000 dilution

Lane 1:

J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell lysate at 10 µg

Lane 2:

Mouse lymph node lysate at 20 µg

Lane 3:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 10 µg

Lane 4:

Mouse placenta lysate at 10 µg

Secondary

Lanes 1 - 3:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lane 4:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 40 kDa

Observed band size: 50-55 kDa

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• WB

CiteAb

Western blot - Anti-CD14 antibody [EPR21847] (AB221678)

CD14 western blot using anti-CD14 antibody [EPR21847] ab221678. Publication image and figure legend from Rao, L., Wu, L., et al., 2020, Nat Commun, PubMed 32999291.

ab221678 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab221678 please see the product overview.

Schematic and characterization of hNVs.a Schematic showing the hNVs consist of engineered SαV-C-NVs, M1-NVs, and P-NVs. b Schematic showing the hNVs efficiently interact with CTCs in the blood, accumulate in the post-surgical tumor bed, repolarize TAMs towards M1 phenotype, and block the CD47-SIRPα 'don't eat me' pathway, thus promoting macrophage phagocytosis of cancer cells, as well as boosting antitumor Tcell immunity. c, d Immunofluorescence imaging c and flow cytometry analysis d of SαV expression on original and engineered B16F10 cells. Scale bar, 10 μm. e Relative mRNA expression of Cd86, Tnf, Il6, and Inos in M0 cells, M1 cells, M0-NVs and M1-NVs. f Hydrodynamic size and zeta potential of P-NVs, M1-NVs, SαV-C-NVs, and hNVs measured by DLS. g TEM images of hNVs. Scale bar, 100 nm and 50 nm in the left and right panel, respectively. The samples were negatively stained with uranyl acetate. h Immunofluorescence images of hNVs. Scale bar, 10 μm. i Western blot analysis of specific protein CD61, CD14, and Melan-A in the samples of P-NVs, M1-NVs, SαV-C-NVs, and hNVs. All data are presented as mean ± S.D. (n = 3). Statistical significance was calculated via unpaired two-tailed t test. **p < 0.01; ***p < 0.001.

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