Native CAB and CAB LAP (200 mg/mL) were graciously obtained from ViiV Healthcare (Research Triangle Park, NC, USA). Myristoyl chloride, poloxamer 407 (P407), N,N-diisopropylethylamine (DIEA), dimethylformamide (DMF), 1-octanal, ciprofloxacin, paraformaldehyde (PFA), and 3,3′-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Corning Life Sciences (Tewksbury, MA, USA). Heat-inactivated pooled human serum was purchased from Innovative Biologics (Herndon, VA, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA). Gentamicin, HPLC grade acetonitrile (ACN), HPLC grade methanol, and Optima grade LC/MS water was purchased from ThermoFisher Scientific (Waltham, MA, USA). FITC mouse anti-human CD45, Alexa Fluor 700 mouse anti-human CD3, APC mouse anti-human CD4, and BV421 mouse anti-human CD8 were purchased from BD Biosciences (San Jose, CA, USA). Monoclonal mouse anti-human HIV-1p24 (clone Kal-1), monoclonal mouse anti-human leukocyte antigen (HLA-DR; clone CR3/43), and the polymer-based HRP-conjugated anti-mouse EnVision + secondary were purchased from Dako (Carpinteria, CA, USA).
Apc mouse anti human cd4
Manufactured by BD
Sourced in United States
The APC mouse anti-human CD4 is a fluorescently-labeled monoclonal antibody that binds to the CD4 surface receptor on human T cells. It is used for the identification and enumeration of CD4+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Fitc mouse anti human cd45, by BD (4 mentions)
Paraformaldehyde pfa, by Merck Group (3 mentions)
Gentamicin, by Thermo Fisher Scientific (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Ciprofloxacin, by Merck Group (3 mentions)
Alexa fluor 700 mouse anti human cd3, by BD (3 mentions)
Bv421 mouse anti human cd8, by BD (3 mentions)
Polymer based hrp conjugated anti mouse envision secondary, by Agilent Technologies (3 mentions)
Myristoyl chloride, by Merck Group (3 mentions)
Fitc mouse anti human cd8, by BD (3 mentions)
Hplc grade methanol, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (2 mentions)
Poloxamer 407 p407 n n diisopropylethylamine diea, by Merck Group (2 mentions)
Monoclonal mouse anti human hiv 1p24 clone kal 1, by Agilent Technologies (2 mentions)
Monoclonal mouse anti human leukocyte antigen hla dr clone cr3 43, by Agilent Technologies (2 mentions)
Hplc grade acetonitrile acn, by Thermo Fisher Scientific (2 mentions)
Optima lc ms grade water, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Pe mouse anti human cd28, by BD (2 mentions)
V450 mouse anti human cd194 ccr4, by BD (2 mentions)
11 protocols using apc mouse anti human cd4
1
Optimizing HIV-1 Virus Detection Assay
2
Immunophenotyping of Human PBMCs
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Human PBMCs were washed with phosphate-buffered saline (PBS) and immunostained with APC mouse anti-human CD4, FITC anti-human ICOS, and PerCP-Cy™5.5-mouse anti-human CD279 (PD-1) (BD Biosciences, San Diego, CA, USA), as well as PE anti-human CD185 (CXCR5) (BioLegend). Isotype-matched antibody controls were used in all procedures; these included FITC mouse IgG1, κ isotype control Alexa, and mouse IgG1 kappa isotype control APC. All the staining procedures were performed according to the manufacturers' protocols, and the stained cells were analyzed using an FACS Calibur flow cytometer and CELLQUEST software (Becton Dickinson, Sparks, MD, USA). The remaining cells were saved in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for real-time polymerase chain reaction (PCR).
3
Multicolor Flow Cytometry Immunophenotyping
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APC Mouse anti-Human CD4 (BD Biosciences), FITC Mouse Anti-Human CD8 (BD), PE Mouse Anti-Human CD28 (BD), V450 Mouse Anti-Human CD194 (CCR4) (BD), Alexa Fluor 700 Mouse anti-Human CD183 (CXCR3) (BD), and PerCP-Cy5.5 Mouse anti-Human CD19 (BD).
4
Multicolor Flow Cytometry of T cells
5
Multicolor Flow Cytometry Immunophenotyping
6
Magnetic Separation of Human CD4+ T Cells
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The peripheral blood mononuclear cell (PBMC) extraction was performed by lymphocytes separation kit (Histopaque 10771, Sigma, USA). Separated PBMC cells from human peripheral blood were labeled with the anti-human CD4+ magnetic particles (Cat. number 557767, BD Bioscience, San Diego, CA, USA). Human CD4+ T lymphocytes were magnetically separated from PBMC on the BD IMagnet (Cat. number 552311, BD Bioscience) according to the instruction protocol. The purity of CD4+ T cells should be more than 95% before next step test. To demonstrate the efficiency of the enrichment and selection steps, cells were stained with APC mouse anti-human CD4 (Cat. number 555349, BD Bioscience) and FITC mouse anti-human CD3 (Cat. number 555332, BD Bioscience). Isotype control was stained by Simultest IgG2a/IgG1 (Cat. number 340394, BD Bioscience). Flow cytometry was performed on a Beckman Coulter FC500 flow cytometry system.
7
Optimizing HIV-1 Virus Detection Assay
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Native CAB and CAB LAP (200 mg/mL) were graciously obtained from ViiV Healthcare (Research Triangle Park, NC, USA). Myristoyl chloride, poloxamer 407 (P407), N,N-diisopropylethylamine (DIEA), dimethylformamide (DMF), 1-octanal, ciprofloxacin, paraformaldehyde (PFA), and 3,3′-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Corning Life Sciences (Tewksbury, MA, USA). Heat-inactivated pooled human serum was purchased from Innovative Biologics (Herndon, VA, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA). Gentamicin, HPLC grade acetonitrile (ACN), HPLC grade methanol, and Optima grade LC/MS water was purchased from ThermoFisher Scientific (Waltham, MA, USA). FITC mouse anti-human CD45, Alexa Fluor 700 mouse anti-human CD3, APC mouse anti-human CD4, and BV421 mouse anti-human CD8 were purchased from BD Biosciences (San Jose, CA, USA). Monoclonal mouse anti-human HIV-1p24 (clone Kal-1), monoclonal mouse anti-human leukocyte antigen (HLA-DR; clone CR3/43), and the polymer-based HRP-conjugated anti-mouse EnVision + secondary were purchased from Dako (Carpinteria, CA, USA).
8
Multicolor Flow Cytometry of T cells
9
Comprehensive Immunophenotyping of CAR-T Cells
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Flow cytometry cell staining was performed at 4°C in PBS supplemented with 2% FBS, unless otherwise indicated. The expression of MSLN on cancer cells was detected by flow cytometry, using a human MSLN PE-conjugated antibody (R&D, FAB32652P, USA).
The expression of CAR on CAR-T cells was detected using biotinylated human MSLN (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA).
The immunophenotypes of T cells were tested using flow cytometry. The antibodies used for analysis include: APC-Cy 7 mouse anti-human CD3 (BD, NJ, USA), APC mouse anti-human CD4 (BD, NJ, USA), phycoerythrin (PE) mouse anti-human CD8 (BD, Franklin Lakes, NJ, USA), Brilliant Violet 421 anti-human CD197 (BioLegend, CA, USA), fluorescein isothiocyanate (FITC) anti-human CD45RA (BioLegend, San Diego, CA, US), PE-Cy7 anti-human CD45RO (BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD197 (CCR7; BioLegend, San Diego, CA, USA), Brilliant Violet 510 anti-human CD62L (BioLegend, San Diego, CA, USA), Brilliant Violet 605 anti-human CD95 (Fas; BioLegend, San Diego, CA, USA), PE anti-human CD223 (LAG-3; BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD366 (Tim-3; BioLegend, San Diego, CA, USA), and APC anti-human CD279 (PD-1; BioLegend, San Diego, CA, USA). Data were analyzed using NovaExpr software (ACEA, Ashland, OR, USA).
The expression of CAR on CAR-T cells was detected using biotinylated human MSLN (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA).
The immunophenotypes of T cells were tested using flow cytometry. The antibodies used for analysis include: APC-Cy 7 mouse anti-human CD3 (BD, NJ, USA), APC mouse anti-human CD4 (BD, NJ, USA), phycoerythrin (PE) mouse anti-human CD8 (BD, Franklin Lakes, NJ, USA), Brilliant Violet 421 anti-human CD197 (BioLegend, CA, USA), fluorescein isothiocyanate (FITC) anti-human CD45RA (BioLegend, San Diego, CA, US), PE-Cy7 anti-human CD45RO (BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD197 (CCR7; BioLegend, San Diego, CA, USA), Brilliant Violet 510 anti-human CD62L (BioLegend, San Diego, CA, USA), Brilliant Violet 605 anti-human CD95 (Fas; BioLegend, San Diego, CA, USA), PE anti-human CD223 (LAG-3; BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD366 (Tim-3; BioLegend, San Diego, CA, USA), and APC anti-human CD279 (PD-1; BioLegend, San Diego, CA, USA). Data were analyzed using NovaExpr software (ACEA, Ashland, OR, USA).
10
Characterization of Nanoformulated HIV Therapy
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DTG was a generous gift from ViiV Healthcare (Research Triangle Park, NC). Pyridine, dimethylformamide (DMF), N,N-diisopropylethylamine (DIEA), myristoyl chloride, poloxamer 407 (P407), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, ciprofloxacin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), and 3,3’-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (St. Louis, MO). Diethyl ether, cell culture grade water (endotoxin-free), gentamicin, acetonitrile (ACN), methanol, KH2PO4, bovine serum albumin (BSA), Triton X-100, LC-MS-grade water, and TRIzol reagent were purchased from Fisher Scientific (Hampton, NH). FITC mouse anti-human CD45, Alexa Fluor 700 mouse anti-human CD3, APC mouse anti-human CD4, BV421 mouse anti-human CD8, PE mouse anti-human CD14, and PE-Cy5 mouse anti-human CD19 were purchased from BD Biosciences (San Jose, CA). Monoclonal mouse anti-human HIV-1p24 (clone Kal-1), monoclonal mouse anti-human leukocyte antigen (HLA-DP/DQ/DR; clone CR3/43), and the polymer-based HRP-conjugated anti-mouse EnVision+ secondary were purchased from Dako (Carpinteria, CA). Heat-inactivated pooled human serum was purchased from Innovative Biologics (Herndon, VA). Dulbecco’s Modification of Eagle’s Medium (DMEM) was purchased from Corning Life Sciences (Tewksbury, MA).
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