PBMC were divided at a 1:10 ratio for enrichment of CD4+ T cells and primary NK cells respectively. Negative-selection strategy was applied using CD4+ T cell or NK-cell enrichment kits (Stemcell Technologies) accoring to the manufacturer’s protocol. Purity of the enriched cell populations was verified by multi-parameter flow cytometry using the following antibodies: CD4-APC (clone RPA-T4), CD3-PacificBlue (clone UCHT1), CD56-APC-Cy7 (clone HCD56), CD8-FITC (clone HIT8a, all BD) and CD16 BV510 (clone EG8, Biolegend). Average cell purity for CD4+ T cells was 97.2% and 94.5% for NK cells respectively with less than 0.3% contamination of CD8+ T cells. CD4+ T cells were resuspended in complete media at 5*106 cells/ml and stimulated with 100 IU/ml human recombinant IL-2 (hrIL-2; NIH) and 1 µg/ml phytohaemagglutinin (PHA, Fisher) overnight. NK cells were resuspended in complete media supplemented with 1 ng/ml human recombinant IL-15 (hrIL-15; R&D systems) overnight.
Cd8 fitc clone hit8a
Manufactured by BD
CD8-FITC (clone HIT8a) is a fluorescently labeled monoclonal antibody that binds to the CD8 surface antigen on T cells. It is a tool used for the identification and enumeration of CD8-positive cells in flow cytometry applications.
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Lab products found in correlation
Cd45ra apc h7 clone 5h9, by BD (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Cd45ro pe cy7 clone uchl1, by BD (1 mentions)
Cd3 apc h7 clone sk7, by BD (1 mentions)
Cd4apc clone rpa t4, by BD (1 mentions)
Cd25 pe clone m a251, by BD (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Cd8 buv395 clone rpa t8, by BD (1 mentions)
Cd4 apc h7 clone l200, by BD (1 mentions)
3 protocols using cd8 fitc clone hit8a
1
Enrichment and Activation of CD4+ T Cells and NK Cells
2
Isolation and Phenotyping of Human Immune Cell Subsets
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Peripheral blood samples (10–20 ml) from 20 healthy young adults (13 F/7 M, age range 22–35 years) were collected in heparinized tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation of whole blood over a Ficoll-Paque gradient (Biochrom) and washed 4× with ice-cold RPMI1640 culture medium (Gibco). CD19+ B cells, CD14+ monocytes, CD3+ T cells, CD4+ CD25− Th cells, CD4+ CD25+ Th cells, CD8+ T cytotoxic cells, CD4+ CD45RA+ CD25− naive Th cells, and CD4CD45RO+ CD25− memory Th cells were isolated by cell sorting using a BD FACS Aria II flow cytometer (BD Biosciences). The sorting strategy is shown in Figure S1 Supplementary Material. The isolated cell populations were phenotyped and used when their purity reached >95%. The antibodies used for cell sorting and phenotyping were the mouse antihuman monoclonal antibodies (mAbs) CD3-APC-H7 (clone SK7), CD19-APC (clone HIB19), CD14-FITC (clone M5E2), CD4-APC (clone RPA-T4), CD8-FITC (clone HIT8a), CD25-PE (clone M-A251), CD45RA-APC-H7 (clone 5H9), and CD45RO-PE-Cy7 (clone UCHL1) (BD Biosciences), CD25-PC5 (clone B1.49.9) (Beckman Coulter). Fluorescence minus one controls were used to identify any background spread of fluorochromes and establish gating boundaries. The data were analyzed using the BD FACS DIVA software v.8.
3
Monoclonal Antibody Staining Panel
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Monoclonal human-reactive mouse IgG2B α-CLEC5A Ab clone #283834, used for functional assays, and the corresponding isotype control (ctrl) clone #20116 were purchased from R&D Systems, USA. For FACS staining, a PE-labelled Ab clone #283834 was used together with its corresponding isotype ctrl, both obtained from R&D Systems. Other monoclonal Abs used in the manuscript for FACS staining are as follows: HLA-DR, DP, DQ BB515 clone Tu39, mouse IgG2ak BB515 clone G155-178, CD83 BUV737 clone HB15e, mouse IgG1k BUV737 clone X40, CD80 APC-R700 clone L307.4, mouse IgG1k APC-R700 clone X40, CD86 BV650 clone 2331, mouse IgG1k BV650 clone X40, CD163 Alexa647 clone GHI/61, mouse IgG1k Alexa647 clone MOP-C21, CD206 BUV395 clone 19.2, mouse IgG1k BUV395 clone X40, CD14 PE clone M5E2, mouse IgG2ak PE clone G155-178, CD274 (PDL1) BB515 clone MIH1, mouse IgG1k BB515 clone X40, TREM1 BV421 clone 6B1, mouse IgG1k BV421 clone X40, CD14 APC-H7 clone MφP9, CD16 BUV395 clone 3G8, CD64 PerCP-Cy5.5 clone 10.1, CD11c BV650 clone B-ly6, CD4 APC-H7 clone L200, CD4 APC clone RPA-T8, CD8 BUV395 clone RPA-T8, CD8 FITC clone HIT8a, CD56 Alexa 488 clone B159, and CD19 BV605 clone SJ25C1 purchased from BD Biosciences, USA. TREM2 PE clone 237820 and Rat IgG2b PE clone 141945 were purchased from R&D Systems.
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