Anti cd45 apc cyanine7

Kidney macrophage isolation at day 14 of AAN was performed via kidney digestion and incubation with Fc Block (catalog No. 14-0161-85, eBioscience, San Diego, CA, USA). Cells were incubated with anti-CD45-APC/Cyanine7 (catalog no. 103115, BioLegend, San Diego, CA, USA), anti-CD11b-FITC (553310, BD, Franklin Lakes, NJ, USA), and anti-F4/80-PE (12-4801-80, eBioscience). CD45⁺ cells were first selected, and CD11b⁺F4/80⁺ cells were then gated for isolation via flow cytometry (BD FACSAria II). Total RNA was extracted from CD45⁺ CD11b⁺F4/80⁺ cells and reverse-transcribed via RT-qPCR analysis, as described below (23 (link)).

RBCs were lysed using appropriate buffer (BD Pharm Lyse™, #555899, BD Biosciences, Franklin Lakes, NJ). Each sample was adjusted to a concentration of 10⁷ WBCs/mL with cold (4°C) FACS buffer (PBS with 2% BSA), and a 100 μL aliquot was placed into a polystyrene tube. Aliquots of stock antibody solutions (20 μL) of anti‐CD45⁺ APC/Cyanine7, anti‐CD3⁺ FITC, anti‐CD4⁺ PE/Cyanine7, anti‐CD8⁺ APC, anti‐CD197(CCR7)⁺ APC/Cyanine7 (BioLegend, San Diego, CA, USA), and anti‐CD14⁺ PE, anti‐CD69⁺ PE, anti‐CD45RA⁺ PE, anti‐CD25⁺ APC‐Cy™7 (BD Biosciences), were added to each sample, and samples were incubated at 4°C for 30 min in the dark. Next, samples were washed (twice) with 2 mL of FACS buffer by centrifugation at 300 × g for 5 min at 4°C. The supernatant was discarded, and labeled cells were re‐suspended in 100 μL of FACS buffer. A 5 μL aliquot of the viability dye (7AAD, BioLegend) was added to each sample and incubated for 10 min at room temperature in the dark. Samples were analyzed using a flow cytometer (Novocyte 2006R, Ace Bioscience, San Diego, CA, USA) with all necessary controls (unstained and compensation).

Tarsal plates from four mice per replicate were pooled in HBSS containing 0.25% trypsin and 50 µg ml⁻¹ DNaseI and incubated at 37 °C for 15 min. After mincing, tissues were dissociated in PBS containing 20% FBS and 0.5 mM EDTA and filtered through a 70-µm strainer. Cell were resuspended into ice-cold PBS containing 2% FBS, 0.5 mM EDTA and 50 µg ml⁻¹ BSA and incubated with anti‐CD45-APC/cyanine7 (BioLegend, 103116, 1:100 dilution) and anti‐Itgav-PE (BioLegend, 104106, 1:100 dilution) for 1 h. Cell sorting was performed using a FACSAria II Flow cytometer (BD Biosciences). Itgav⁺;CD45⁻ meibomian gland cells (approximately 10,000 cells per replicate) were sorted into DMEM/Ham’s F12 medium containing 10% FBS. After sorting, cells were pelleted and total RNA was directly extracted using an RNeasy micro kit (QIAGEN). In our study, CD45 expression was used to exclude hematopoietic cells from the analysis.