Carboxyfluorescein succinimidyl ester (cfse)

Neutrophils obtained after 4-day differentiation (“in vitro-derived”) were washed and labeled using CFSE (BioLegend). Murine bone marrow neutrophils were isolated by negative selection (StemCell Technologies) and immediately labeled alongside in vitro-derived neutrophils using CFSE. 96-well plates were coated for 1 hour at room temperature or overnight at 4 °C with 2.5, 5, or 7.5 µg/mL ICAM-1 and/or 2.5 µg/mL CXCL1 in phosphate buffered saline (PBS) and then blocked with 1% casein (ThermoFisher) or 0.5% polyvinylpyrrolidone (Sigma Aldrich) in PBS for 2 hours at room temperature. Neutrophils were loaded into the 96-well plate at 0.5 × 10⁶ neutrophils per well in Hank’s balanced salt solution containing Ca²⁺ and Mg²⁺ (HBSS⁺⁺), and then incubated at 37 °C for 35 or 65 minutes. Following incubation, neutrophils were quantified by a plate reader for fluorescence intensity (CFSE), before and after sequential gentle washes with HBSS⁺⁺. The number of adherent neutrophils was inspected visually by light microscopy to corroborate with plate reader signal intensity. Each group was replicated in three to six wells per independent experiment.

Murine
bone-marrow-derived dendritic cells (BMDCs) (5 × 10⁵ cells/mL) from BALB/c mice were pretreated with the vehicle
or broad Ca²⁺ channel blocker (SKF96365, 20 μM) and
treated with vehicle 2D216 or its derivatives (5 μM)
in combination with MPLA (100 ng/mL) for 24 h. BMDCs were loaded with
the OVA protein (10 μg/mL) for 4 h, washed twice, and co-cultured
with the same number of CFSE-labeled CD4 T cells from spleens of sex-matched
DO11.10 TCR transgenic mice for 72 h. CD4⁺ T cells were
isolated from spleens of DO11.10 TCR transgenic mice using an EasySep
Mouse CD4 T cell isolation kit (STEMCELL Technologies, Vancouver,
Canada) and labeled with CFSE (4 μM, Molecular Probe, Eugene,
OR, United States). Supernatants were assayed for IFN-γ and
IL-2 by ELISA, and cell suspensions were subjected to fluorescence-activated
cell sorting (FACS) analysis of CFSE dilution of DO11.10 CD4 T cells.
T cell division was analyzed by a MACSQuant Analyzer 10 (Miltenyi
Biotec, Bergisch Gladbach, Germany) using AF647-conjugated anti-DO11.10
TCR antibodies (eBioscience, San Diego, CA, United States). The %
divided, the percent of the live CFSE-labeled CD4⁺ T cells
that entered division, was calculated using FlowJo (version 10.6.1,
Becton Dickinson, Ashland, OR, United States).

The carboxyfluorescein diacetate succinimidyl ester (CFSE, Biolegend, USA) was used to label the proliferation of the PBMCs. The cells were resuspended with 5 μM CFSE working fluid and incubated in a carbon dioxide cell incubator for 20 min. Next, a five-time volume of the RPIM1640 medium containing 10% fetal bovine serum was added to terminate the reaction, and then washed three times by RPIM1640 medium. After labeling with CFSE, the cells were incubated with human CD3/CD28 T cell activator (Stem Cell, Canada) at a dose of 25 µL/mL and IL-2 (Pepro Tech, USA) at a dose of 50 ng/mL for up to 3 d, and then analyzed by fluorescence-activated cell sorting (FACS).