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Anti cd19

Manufactured by Cell Signaling Technology
Sourced in United States

Anti-CD19 is a monoclonal antibody targeting the CD19 cell surface antigen. CD19 is a transmembrane glycoprotein that is expressed on the surface of B cells and their precursors. Anti-CD19 can be used for the identification and analysis of B cells in various applications.

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5 protocols using anti cd19

1

Fluorescent Protein and Antibody Labeling Protocol

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GFP- and tdTomato-tagged F-Tractin were gifts from Michael Schell (Uniformed Services University, Maryland). Alexa Fluor-conjugated phalloidins were purchased from Thermo Fisher. Anti-mDia1 antibody was purchased from Thermo Fisher (PA5-27607). HRP-conjugated mouse anti-β-actin antibody was purchased from Santa Cruz (SC-47778 HRP). Rabbit anti-CD79a (#3351), anti-PCD79a (#5173), anti-CD19 (#3574), and anti-PCD19 (#3571) were purchased from Cell Signaling Technologies. Anti-M2A was purchased from MilliporeSigma (#M8064). CK-666 and SMIFH2 were purchased from MilliporeSigma and used at final concentrations of 100 μM and 25 μM, respectively. pnBB and paBB were purchased from Cayman Chemicals and used at a final concentration of 25 μM. DMSO vehicle control was purchased from MilliporeSigma. CellMask Deep Red Plasma Membrane Stain was purchased from Thermo Fisher. Alexa Fluor 488- (#111-545-003), 594- (#111-585-003), and 647- (#111-605-003) conjugated goat, anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch. Goat anti-mouse IgG Fcγ fragment-specific antibody (#115-005-008) and goat anti-mouse IgM, µ-chain-specific antibodies (#115-005-020) were purchased from Jackson ImmunoResearch. Anti-rabbit-HRP (#32260) was purchased from Thermo Fisher.
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2

Immune Signaling Profiling in COVID-19 Recovery

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PBMCs from COVID-19 recovered patients and healthy donors were incubated with 10 μg/ml biotin-F(ab′)2 anti-human Ig(M + G) on ice for 30 min, plus 20 μg/ml streptavidin on ice for 10 min, and then activated at 37 °C for indicated times. Cell lysates were used for electrophoresis in SDS–polyacrylamide gel, electrotransferred onto a nitrocellulose membrane, and then probed with the following specific antibodies: anti-pCD19 (Cat# 3571S, Cell Signaling Technology, USA), anti-CD19 (Cat# 90176, Cell Signaling Technology, USA), anti-pBtk (Cat# ab52192, Abcam, USA), anti-Btk (Cat# 8547S, Cell Signaling Technology, USA), anti-pPI3K (Cat# 4228S, Cell Signaling Technology, USA), anti-pAkt (Cat# 4060L, Cell Signaling Technology, USA), anti-pFoxO1 (Cat# 9461S, Cell Signaling Technology, USA), anti-pmTOR (Cat# 5536S, Cell Signaling Technology, USA), and anti-pS6 (Cat# 4856S, Cell Signaling Technology, USA). After incubated relative secondary antibodies, immunoreactive bands were presented and captured with the ChemiDoc XRS + imaging systems (Bio-Rad). β-actin or GAPDH was used as the loading control.
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3

Comprehensive Antibody Detection Protocol

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The following antibodies were used for protein detection: anti-CD19–PE (Beckman Coulter; IM1285U), anti-CD19 (Cell Signaling; 3574), anti-CD19 3G7 (Origen; TA506234), anti-CD19 HD37 (Thermo Fisher; MA1-34005), anti-VSVg–fluorescein isothiocyanate (FITC) (Abcam; ab3863), anti-VSVg (Abcam; ab50549), anti-CD81 (LifeSpan; LS-C359239), anti-CD81–FITC (Molecular Probes; A15753), anticalnexin (Cell Signaling; 2679), anticalreticulin (Cell Signaling; 12238), anti-UGCGL1 (NovoPro Laboratories; 116554), anti-PDI (Cell Signaling; 3501), anti-mouse antibody–Alexa Fluor 488 (Thermo Fisher; R37114), and anti-rabbit antibody–Alexa Fluor 594 (Thermo Fisher; A-21207).
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4

Double Immunofluorescence Staining of B-Cells

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Double immunofluorescence staining was performed as previously described.36 (link) Briefly, B-cells were fixed in 4% paraformaldehyde (15 min, RT), washed with PBS, permeabilized (0.1% Triton X-100 in PBS, 15 min), and attached on pre-coated poly-L-lysine slides. Slides were blocked in 3% BSA, followed by mouse monoclonal anti-NS3 (Abcam; 1:100, ab65407) or anti-core (ThermoFisher Scientific, Waltham, MA, USA; 1:100, MA1-080) antibody staining. The cells were further incubated with rabbit polyclonal anti-CD19, anti-CD20 (Cell Signaling Technology, Inc., Danvers, MA, USA; 1:400; 3574), anti-CD20 (Abcam; 1:400; ab78237) or anti-B220 antibody (Biolegend, San Diego, CA, USA; 1:200; #103201). Cells were washed with PBS and incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse or anti-rat Alexa Fluor 597 (Invitrogen; 1:200). The stained slides were mounted using Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA, USA). Control slides were similarly processed, except primary antisera were omitted, which yielded no staining. Images were captured using an Olympus FV1000 confocal microscope and processed with Olympus Fluoview Version 1.7c software (Olympus Corporation, Tokyo, Japan).
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5

IHC Staining of Mouse Tissue Sections

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Mouse tissues were fixed in 10% neutral buffered formalin (Sigma) and embedded in paraffin, then sectioned (5 μm) onto glass slides. Slides were deparaffinized and rehydrated using a standard histology protocol. Antigen retrieval was performed using 10 mM sodium citrate pH 6.0 buffer and a Decloaking chamber at 120°C. The slides were stained using an Autostainer Plus (Dako) with TBST rinse buffer (Dako). The following antibodies were used for IHC: anti-CD4 (Cell Signaling, D7D2Z, 1:100), anti-CD8 (Cell Signaling, D4W2Z, 1:400), anti-CD3 (Biocare Medical, 1:100), and anti-CD19 (Cell Signaling, D4V4B, 1:800). Detection consisted of Rabbit Boost (Cell Signaling) HRP polymer used with 3,3’-diaminobenzidine (DAB) (Dako). Slides were finally counterstained with hematoxylin (Dako). Images were captured with an Olympus AX70 microscope using the Q-Capture Pro7 Program. H&E stained sections and immunohistochemistry of lymphoid malignancies and polycystic changes were evaluated by two board-certified pathologists including a veterinary pathologist (LHR).
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