Six (FITC) tagged MoA againstFoxp3 and CD14, phycoerythrin

Six (6)ml ofvenousbloodwasdrawn aseptically from anterior cubital veinand addedinto3vacutainers, 2 ml in an EDTA vial  for total leucocytescount and differential leucocytes count, 2 ml in clot activator vial for HIVtesting and 2 ml in heparin vials for immunophenotyping and IGRA test. All sampleswereprocessed withinfourhoursand analyzedon thesameday.

Immunophenotypicanalysis:  Twenty microliter of each of APC-Cy7 taggedmonoclonal antibody against( MoA)  CD4, Fluoresceinisothiocyanate(FITC) taggedMoAagainstFoxp3and CD14, phycoerythrinCy7(PE-Cy7) tagged MoA against PD1,APC tagged MoA against PDL1 and PE-Cy5 tagged MoA against CD25 were used forstaning . All theseantibodies were purchasedfrom BDBioscience (San Diego, CA,USA). Two panels were used.

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One panel (panel-1)was used for CD4-APC-Cy7 ,PD1-PE-Cy7, CD25-PECy5 and  Foxp3-FITC. One panel( panel-2) used for  CD4-APC-Cy7, PDL1-APC, PD1-PE-Cy7 and CD14-FITC. Lyse-washsamplepreparationmethod usingwhole blood was performed.For staining 100 ?l ofwhole blood ofeach subject was placed into2 FACStubes foreach panel.One tube used for stainingand another for FMO control ( Fluorescence –minu-one). Gating of surface andintracellular markers were determined using control smples by the  FMO approach i.e.

controls containing allmarkers except the one of interest were used to set gates.  For compensation control, BD FACSTM 7-colorsetup beads along with  BD Diva softwarewere used. The sofware automatically calculate the accurate compensation value.      Tubes were mixedand incubatedin dark atroom temperature for15minutes.

Twoml ofBDFACSLyse (<15%formaldehydeand<50%diethyleneglycol,wasdiluted1:10in deionisedwater immediately before use)wasadded toeach tube. Tubes were re-incubated indarkf or10 minutes. Centrifuged at1200 rpm for10minutesand supernatant was discarded. Pellet was brokenand cells werewashedtwiceby adding2ml of sheathfluid, mixed, centrifuged andsupernatant was discarded.

Cellswerere-suspended in0.5ml ofsheath fluid with 2%paraformaldehyde.For intracellular staining 0.

5 mlof 1X BD permiabilization buffer was used and keep it for 30 min and centrifugedat1200rpm for 5 minutesand supernatant was discarded. Pellet was brokenand 20 ?L of Foxp3-FITC monoclonal antibodies was added and incubate at roomtemperature for 30 min. The tubes were washed twice byadding 2mlof sheathfluid, mixed, centrifuged andsupernatant was discarded. Cells were re- suspendedin 0.5 mlof sheathfluidwith2%  paraformaldehyde.Flowcytometryanalysis was done on BD FACS Canto-II.

The percentage of positive cells and themedian fluorescent intensity (arbitrary units) for a specific marker wascalculated by using BD FACS Diva software. For analysis 50000 events wererecorded.