Six (FITC) tagged MoA againstFoxp3 and CD14, phycoerythrin

Six (6)ml of
venous
blood
was
drawn aseptically from anterior cubital vein
and added
into
3vacutainers, 2 ml in an 
EDTA vial  for total leucocytes
count and differential leucocytes count, 2 ml in clot activator vial for HIV
testing and 2 ml in heparin vials for immunophenotyping and IGRA 
test. All samples
were
processed within
four
hours
and analyzed
on the
same
day.

Immunophenotypic
analysis:

  Twenty microliter of each of APC-Cy7 tagged
monoclonal antibody against( MoA)  CD4, Fluoresceinisothiocyanate
(FITC) tagged
MoA
againstFoxp3
and CD14, phycoerythrin
Cy7(PE-Cy7) tagged MoA against PD1,
APC tagged MoA against PDL1 and PE-Cy5 tagged MoA against CD25 were used for
staning . All these
antibodies were purchased
from BD
Bioscience (San Diego, CA,USA). Two panels were used. 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-wash
sample
preparation
method using
whole blood was performed.
For staining 
100 ?l of
whole blood of
each subject was placed into
2 FACS
tubes for
each panel.One tube used for staining
and another for FMO control ( Fluorescence –minu-one). Gating of surface and
intracellular markers were determined using control smples by the  FMO approach i.e. controls containing all
markers except the one of interest were used to set gates.  For compensation control, BD FACSTM 7-color
setup beads along with  BD Diva software
were used. The sofware automatically calculate the accurate compensation value.

      Tubes were mixed
and incubated
in dark at
room temperature for15minutes.Two
ml of
BDFACSLyse (<15%formaldehyde and<50%diethylene glycol, was diluted1:10 in deionised water immediately before use) was added to each tube. Tubes were re-incubated in dark f or10 minutes. Centrifuged at1200 rpm for10minutes and supernatant was discarded. Pellet was broken and cells were washed twice by adding 2ml of sheath fluid, mixed, centrifuged and supernatant was discarded.Cells were re-suspended in 0.5 ml of sheath fluid with 2% paraformaldehyde. For intracellular staining 0.5 ml of 1X BD permiabilization buffer was used and keep it for 30 min and centrifuged at1200 rpm for 5 minutes and supernatant was discarded. Pellet was broken and 20 ?L of Foxp3-FITC monoclonal antibodies was added and incubate at room temperature for 30 min. The tubes were washed twice by adding 2ml of sheath fluid, mixed, centrifuged and supernatant was discarded. Cells were re- suspended in 0.5 ml of sheath fluid with 2%  paraformaldehyde. Flowcytometry analysis was done on BD FACS Canto-II. The percentage of positive cells and the median fluorescent intensity (arbitrary units) for a specific marker was calculated by using BD FACS Diva software. For analysis 50000 events were recorded.