Foxp3 fix perm kit protocol

Single cell suspensions of leukocytes from blood, ascites, tumor or omental metastases were stained with the following monoclonal antibodies: anti-CD3 APC-H7 (BD Pharmingen, 560176), anti-CD4 eFlour 450 (eBioscience, 48-0048-42), anti-CD8 Pacific Orange (Invitrogen, MHCD0830), anti-CD25 APC (BD Pharmingen, 555434), or anti-CTLA-4 APC (BD Pharmingen, 555855), anti-CD28 PerCpCy5.5 (eBioscience, 45-0289-42), anti-CD38 PE-TR (Invitrogen, MHCD3817), anti-HLA-DR PE-Cy7 (BD Biosciences, 335795), anti-FoxP3 PE (eBioscience, 12-4777-42), anti-ki67 FITC (BD Pharmingen, 556026) or CD39 FITC (BD Pharmingen, 561444) or anti-Helios (Biolegend, 137214). Cells were stained in FACs buffer (1%FBS in PBS with 0.01%NaN₃) and fixed according to the ebioscience FoxP3 Fix-Perm kit protocol (eBioscience, 00-5521-00). All samples were run on a BD LSRII Flow cytometer and analysed by FACSDiva BD. Briefly, for every single flow cytometric antibody, we have used Fluorescent Minus One (FMO), to discriminate between positive and negative cells [35 (link)].

Single cell suspensions of leukocytes from peripheral blood were stained using the following monoclonal antibodies: anti-CD3 APC-H7 (BD, 641397), anti-CD4 Alexa Fluor 700 (BD, 557922), anti-CD8 Alexa Fluor 700 (BD, 557945), anti-CD25 APC (Miltenyi 130-092-858), anti-CD28 BV421(BD, 562613), anti-CD38 BV605 (Biolegend 303532), anti-HLA-DR PE-Cy7 (BD Biosciences, 335795), anti-CD39 BV421 (BD 563679), anti-CD45 RO BV510 (BD, 563215), anti-CD56 APC (Biolegend 318310), anti-CCR7 PE (BD 561008), anti-FoxP3 PE (eBioscience, 12-4777-42), anti-Ki-67 FITC (BD 556026), anti-HELIOS (Biolegend, 137214), anti-CTLA-4 APC (BD 560938), anti-OX40 FITC (Biolegend 35006), and anti-PD1 BV510 (Biolegend 329932). Cells were stained in FACs buffer (1 % FBS in PBS with 0.01 % NaN₃) and fixed according to the eBioscience FoxP3 Fix-Perm kit protocol (eBioscience, 00-5521-00). All samples were analyzed on a BD X20 Fortessa Flow cytometer. We used Fluorescence Minus One to discriminate between positive and negative cells for each antibody [33 (link)].