Anti cd62l percp cy5

Splenocytes or lung cells were prepared and counted from each mice in different groups as previously described, respectively [15 (link), 16 (link)]. Cells were plated in triplicate at 5 × 10⁶ cells per well in a 24-well plate and incubated with CMFO (10 μg/mL) and anti-CD28/CD49d (1 μg/mL, eBioscience) overnight. RPMI 1640 medium used is a negative control. Cell responses were monitors through a cell stimulation cocktail (1 μg/mL, eBioscience). Cells were stained for anti-CD4-APC-Cy7 (Cat#552051, BD Biosciences), anti-CD8α-BV510 (Cat#563068, BD Biosciences), anti-CD44-FITC (Cat#561859, BD Biosciences), anti-CD62L-PerCP-Cy5.5 (Cat#560513, BD Biosciences), and intracellular markers anti-IFN-γ-PE (Cat#554412, BD Biosciences) and anti-IL-2-APC (Cat#554429, BD Biosciences). Absolute number of IFN-γ⁺ and IL-2⁺ T cells, central memory T cells (T_CM), and effector memory T cells (T_EM) was determined by an LSRII multicolor flow cytometer (BD Biosciences) and analyzed through FlowJo software. The results were shown as the mean ± SEM per group (n = 6).

Monocyte-derived macrophages or BMDMs were harvested and washed in cold PBS. Cells were incubated in Fc-block reagent (BD biosciences) and fixable viability dye eFluor 780 (Invitrogen) for 15 min at 4°C in cold PBS. Monocyte-derived macrophages were washed once and stained with the following antibodies: anti-CD62L PerCp-Cy5.5 (BD biosciences), anti-CCR2 APC (R and D Systems), anti-F4/80 efluor450 (eBioscience), anti-CSF1R BV711 (Biolegend), anti-Ly6G BUV395 (BD biosciences), anti-CD11b BUV737 (BD biosciences), anti-MHCII(I-A/I-E) PE (eBioscience) and anti-Ly6C PE-Cy7 (eBioscience). BMDMs were washed once and stained with the following antibodies: anti-CD45 FITC (eBioscience), anti-F4/80 BV421 (Biolegend), anti-CD11b BUV395 (BD biosciences). Stained cells were analyzed on by flow cytometry using a BD FACS CANTO instrument. Loss of eGFP or CD45 was assessed by gating on live F4/80 + macrophages using FlowJo X.

Single cell suspensions were made from the spleen and draining lymph nodes, red blood cells were lysed using an ammonium chloride buffer (Sigma Aldrich, Dorset, UK), and cells were then re-suspended in FACS buffer (PBS, 2% fetal calf serum, 0.01% sodium azide (Sigma Aldrich, Dorset, UK). Fc receptors were blocked with supernatant from the hybridoma 2.4G2. All antibodies were from eBioscience, Hatfield, UK, except where stated; live/dead fixable cell stain conjugated to ef455 (Life Technologies), anti-CD4-APC, anti-CD4-AF700 (BD Pharmingen, Oxford, UK), anti-CD11c-PE-Cy7, anti-CD11c-ef450, anti-Ki67-PE-Cy7, anti-CD11b-Af700, anti-CD45.1-FITC, anti-CD44-APC-Cy7, anti-CD80-PE, anti-CD86-APC, anti-CD62L-PerCP-Cy5.5, and anti-Foxp3-ef450. FACS data were collected using a 6 laser LSR Fortessa (BD Biosciences, New Jersey, USA) and analyzed using FlowJo software (Tree Star, Olten, Switzerland).