After blood collect, erythrocytes were lysed by using a commercially available hypotonic lysis solution (BD FACSTM, BD Biosciences, San Jose, CA, USA), leukocytes obtained after centrifugation (600x g, 10 mins) were resuspended in Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific) added 0.1% bovine serum albumin (BSA, Sigma-Aldrich), and incubated with anti-L-selectin fluorescein-conjugated (10 µL/106 cells, Bio-Techne, Minneapolis, MN, USA) or anti-ICAM-1 Alexa Fluor® 488-conjugated monoclonal antibody (1 µg, Santa Cruz Biotechnology, Dallas, TX, USA) for 20 min, at 4ºC. Negative control was not added any antibody. Leukocytes were washed with HBSS plus BSA buffer, fixed in 4% paraformaldehyde solution for 10 min, and fluorescence was measured 12 h later in a flow cytometer (BD Accuri C6, BD Biosciences, San Jose, CA, USA). Ten thousand cells were counted per sample.
Facs
Manufactured by BD
Sourced in United States
The BD FACSTM is a flow cytometry instrument designed for cell analysis and sorting. It provides high-performance detection and measurements of various cellular parameters, including size, granularity, and fluorescence intensity.
Automatically generated - may contain errors
2 protocols using facs
1
Leukocyte Surface Marker Analysis
2
Placental Immune Cell Isolation
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Cells were isolated from each placenta as previously described [19] (link) and illustrated in Supplementary Figure 1. Placentae were collected in RPMI 1640 supplemented with 10% fetal calf serum and penicillin/streptomycin at room temperature and processing was performed within six hours of delivery. Each placenta was dissected to obtain the decidua parietalis, basalis and villous tissue. Enzymatic lymphocyte isolation was performed using Collagenase I and DNAse I. The lymphocyte fraction was obtained following Percoll density centrifugation and incubated with violet amine reactive viability dye (VIVID, Thermofisher). The cells were then fixed using BD FACS TM lysing solution and cryopreserved in liquid nitrogen until analysis. Cord blood mononuclear cells were isolated on Ficoll, fixed and cryopreserved until analysis.
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