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Cd4 apc clone okt4

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CD4-APC clone OKT4 is a fluorescent-labeled monoclonal antibody used to identify and quantify CD4-positive cells in flow cytometry applications. It targets the CD4 antigen, which is expressed on the surface of T helper cells.

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Lab products found in correlation

Cd3 percp clone sk7, by BioLegend (2 mentions) Cd8 bv570 clone rpa t8, by BioLegend (2 mentions) Cd14 fitc clone 63d3, by BioLegend (2 mentions) Cd19 bv510 clone sj25c1, by BD (2 mentions) Ghost dye a710 viability stain, by Cytek Biosciences (2 mentions) Facsaria fusion instrument, by BD (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (2 mentions) Cd3 af700 clone okt3, by BioLegend (2 mentions) Cd8a pe clone rpa t8, by BioLegend (2 mentions) Sfemii, by STEMCELL (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Anti cd3 clone hit3a, by BioLegend (1 mentions) Anti cd28 clone cd28, by BioLegend (1 mentions) Clone hit3a, by BioLegend (1 mentions) Carboxyfluorescein diacetate succinimidyl ester cfse, by Thermo Fisher Scientific (1 mentions) Clone cd28, by BioLegend (1 mentions) Aria 2 fluorescence activated cell sorter, by BD (1 mentions) Anti human cd66b percpcy5.5 clone g10f5, by BioLegend (1 mentions)

Close spelling variants of this product

CD4 (clone OKT4; CD4 (OKT4, CD4-APC

5 protocols using cd4 apc clone okt4

1

Detailed PBMC Immunophenotyping Protocol

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1M PBMCs from each donor were stained using standard procedure (30 min, 4 C) with the following surface antibody panel (CD3-PerCP clone SK7 (BioLegend), CD4-APC clone OKT4 (BioLegend), CD8-BV570 clone RPA-T8 (BioLegend), CD14-FITC clone 63D3 (BioLegend), CD19-BV510 clone SJ25C1 (BD), and Ghost dye A710 viability stain (Tonbo)) (Life Sciences Reporting Summary). Samples were then analyzed and sorted using a BD FACSAria Fusion instrument at the UCSF flow cytometry core. To calculate cell type proportions, the number of events in each of CD3+ CD4+ CD8− (CD4+ T cells), CD3+ CD4− CD8+ (CD8+ T cells), CD3− CD19+ (B cells), and CD3− CD14+ (monocytes) were divided by the sum of events in these gates (Supplementary Fig. 21).
Kang H.M., Subramaniam M., Targ S., Nguyen M., Maliskova L., McCarthy E., Wan E., Wong S., Byrnes L., Lanata C., Gate R., Mostafavi S., Marson A., Zaitlen N., Criswell L.A, & Ye C.J. (2017). Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nature biotechnology, 36(1), 89-94.
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2

T Cell Co-Culture with Immune Cells

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T cell co-culture was performed as previously described (78 (link)) with minor modifications. Briefly, tissue-culture plates were coated with 5 μg/mL purified anti-CD3 (clone HIT3a, Biolegend) at 4°C overnight and subsequently washed twice with 1X PBS. PBMCs from a healthy donor (Research Blood Components) were labelled with CFSE (Invitrogen) following the manufacturer’s protocol. PBMCs were resuspended in SFEM II (StemCell Technologies) with 5 μg/mL purified anti-CD28 (clone CD28.2, BioLegend) and plated at a density of 1M cells/mL. Isolated iMS1 or iMono cells were added at different ratios as indicated. The cells were left in culture for 3–4 days, with media replenished after 2 days. At the end of incubation, the cells were stained with CD3-AF700 (clone OKT3), CD4-APC (clone OKT4), and CD8a-PE (clone RPA-T8) (BioLegend) to determine the amount of CFSE dilution within the T cells.
Reyes M., Filbin M.R., Bhattacharyya R.P., Sonny A., Mehta A., Billman K., Kays K.R., Pinilla-Vera M., Benson M.E., Cosimi L.A., Hung D.T., Levy B.D., Villani A.C., Sade-Feldman M., Baron R.M., Goldberg M.B., Blainey P.C, & Hacohen N. (2021). Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro. Science Translational Medicine, 13(598), eabe9599.
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3

T Cell Proliferation Assay with iMS1 and iMono

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T cell coculture was performed as previously described (78 (link)) with minor modifications. Briefly, tissue culture plates were coated with purified anti-CD3 (5 μg/ml) (clone HIT3a, BioLegend) at 4°C overnight and subsequently washed twice with 1X PBS. PBMCs from a healthy donor (Research Blood Components) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) following the manufacturer’s protocol. PBMCs were resuspended in SFEM II (StemCell Technologies) with purified anti-CD28 (5 μg/ml) (clone CD28.2, BioLegend) and plated at a density of 1 million cells/ml. Isolated iMS1 or iMono cells were added at different ratios as indicated. The cells were left in culture for 3 to 4 days, with media replenished after 2 days. At the end of incubation, the cells were stained with CD3-AF700 (clone OKT3), CD4-APC (clone OKT4), and CD8a-PE (clone RPA-T8) (BioLegend) to determine the amount of CFSE dilution within the T cells.
Reyes M., Filbin M.R., Bhattacharyya R.P., Sonny A., Mehta A., Billman K., Kays K.R., Pinilla-Vera M., Benson M.E., Cosimi L.A., Hung D.T., Levy B.D., Villani A.C., Sade-Feldman M., Baron R.M., Goldberg M.B., Blainey P.C, & Hacohen N. (2021). Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro. Science Translational Medicine, 13(598), eabe9599.
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4

Detailed PBMC Immunophenotyping Protocol

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1M PBMCs from each donor were stained using standard procedure (30 min, 4 C) with the following surface antibody panel (CD3-PerCP clone SK7 (BioLegend), CD4-APC clone OKT4 (BioLegend), CD8-BV570 clone RPA-T8 (BioLegend), CD14-FITC clone 63D3 (BioLegend), CD19-BV510 clone SJ25C1 (BD), and Ghost dye A710 viability stain (Tonbo)) (Life Sciences Reporting Summary). Samples were then analyzed and sorted using a BD FACSAria Fusion instrument at the UCSF flow cytometry core. To calculate cell type proportions, the number of events in each of CD3+ CD4+ CD8− (CD4+ T cells), CD3+ CD4− CD8+ (CD8+ T cells), CD3− CD19+ (B cells), and CD3− CD14+ (monocytes) were divided by the sum of events in these gates (Supplementary Fig. 21).
Kang H.M., Subramaniam M., Targ S., Nguyen M., Maliskova L., McCarthy E., Wan E., Wong S., Byrnes L., Lanata C., Gate R., Mostafavi S., Marson A., Zaitlen N., Criswell L.A, & Ye C.J. (2017). Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nature biotechnology, 36(1), 89-94.
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5

MDSC Isolation and Characterization

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Peripheral blood was collected in sterile heparinized tubes from each patient, and PBMC, obtained by Ficoll–Hypaque gradient centrifugation, were analyzed by flow cytometry. The following anti-human fluorescence conjugated antibodies and their corresponding isotype controls used were as follows: anti-human CD11b-APC-cy7 (clone ICRF44, BD), anti-human HLA-DR-PE (clone G46-6, BD), anti-human CD14-FITC (clone 63D3, Biolegend), and anti-human CD66b-Percpcy5.5 (clone G10F5, Biolegend), FVD (BD), CD3-PE-cy7 (clone HIT3a, Biolegend), CD4-APC (clone OKT4, Biolegend), CD8-PB (clone RPA-T8, Biolegend). We used FVD to discriminate dead cells, and MDSC were classified into two subsets, G-MDSC (CD11b+ HLA-DRlow/− CD66b+) and M-MDSC (CD11b+ HLA-DRlow/CD14+). Data were analyzed with FlowJo 10.0 software package (Treestar Inc., Ashland, OR, USA).
For flow cytometric sorting, an Aria II fluorescence activated cell sorter (BD, Mountain View, CA, USA) was used. The strategy for MDSC sorting was CD11b+ HLA-DRlow/− cells from live PBMC. Depletion of MDSC was performed by harvesting the remaining PBMC after MDSC sorting.
Hu C., Zhen Y., Pang B., Lin X, & Yi H. (2019). Myeloid-Derived Suppressor Cells Are Regulated by Estradiol and Are a Predictive Marker for IVF Outcome. Frontiers in Endocrinology, 10, 521.
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