Carboxyfluorescein succinimidyl ester (cfse)

C57BL/6 J wild-type (Stock# 000664), μMT (Stock# 002288), and CD45.1 congenic (Stock# 002014) mice were purchased from Jackson Labs. Blimp-1-YFP mice were a gift of Dr. Sue Kaech^{42 (link)}. All experimental mice were between 6 and 9 weeks of age. CD43⁻ splenic B cells were purified from naive mice by magnetic purification according to the manufacturer’s instructions (Miltenyi Biotech). To induce B cell differentiation, 50 μg LPS (Enzo Life Sciences, ALX-581-008) was i.v. injected into naive mice and three days later CD138⁺ splenic plasmablasts were isolated by FACS^{2 (link)}. For division labeling, purified B cells were stained with 2 μM CFSE (Tonbo Biosciences) for 8 min or 5 μM CTV (ThermoFisher Scientific) for 20 min at room temperature protected from light. CFSE staining was quenched with 1 volume FBS, and for both dyes, cells were washed with 5 volumes B cell media (RPMI 1640, 10% heat-inactivated FBS, 10 mM HEPES, 1 mM sodium pyruvate, 1× non-essential amino acids, 1× PeneStrep, 0.05 mM 2-ME), and either cultured or adoptively transferred into μMT hosts. For in vivo LPS challenge, one day post adoptive transfer mice were injected i.v. with 50 μg LPS as above. All animal experiments were approved by the Emory University Institutional Animal Care and Use Committee.

To visualize binding and phagocytosis of bacteria by neutrophils, neutrophils (isolated from separate donors) were diluted to 2.0 × 10⁶ cells/ml and seeded onto tissue culture-treated glass coverslips with 1% HSA or 1% NHS (45 (link), 46 (link)). Bacteria were resuspended to an OD₆₀₀ of ∼0.8 in PBS, stained with 5 μg/ml CFSE (Tonbo Biosciences, San Diego, CA) for 20 min at 37°C, and washed twice with PBS. CFSE-stained bacteria were added at an MOI of 10:1 and centrifuged at 1,000 rpm for 2 min. Samples were incubated for 30 min at 37°C with 5% CO₂. Cells were washed twice with PBS to remove nonadherent bacteria and were fixed at RT for 15 min using 4% paraformaldehyde in PBS. Samples were incubated with anti-K. kingae antibody (1:500) overnight at 4°C. The anti-K. kingae antibody was generated against an acetone powder of K. kingae strain KK01 whole bacteria. Cells were washed twice with PBS and incubated with a 1:500 dilution of goat anti-guinea pig IgG DyLight 649-conjugated antibody (Rockland, Limerick, PA).
Visualization of bacteria-neutrophil association was performed by microscopy using a Nikon Eclipse Ni-E (Nikon Instruments, Inc., Melville, NY) with either a 20× objective or 60× oil objective. For quantification, 100 neutrophils per replicate were chosen at random for analysis; the cell-associated and extracellular bacteria were counted by a blinded reader.

For proliferation assays, cells were labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Tonbo Biosciences) for 8 min at 37°C and seeded at progressively reduced density (no less than 3 000 cells/cm²). After recovery for 24 h, samples were collected at 24-h intervals and fixed in 70% ice-cold ethanol. CFSE fluorescence intensity was recorded for 10 000 single-cell events using a BDAccuri C6 flow cytometer (BD Biosciences). The mean of fluorescence intensity (MFI) of the green FL1-A channel (excitation 488 nm, emission 530 ± 15 nm) was calculated using FlowJo software. Relative growth for each time point was calculated by normalizing MFI^–1 of individual time points to the MFI^–1 value at 24 h post-seeding.

A 24-well plate was coated with 1 μg/well of the anti-CD3e antibody. CD8⁺ T cells were purified from the spleen of a Balb/c mouse using T-cell isolation columns (R&D systems, Minneapolis, MN, USA) according to the manufacturer's instructions and labeled with carboxyfluorescein succinimidyl ester (CFSE). In order to assess the impact of MDSC on CD8⁺ T cell proliferation, CD8⁺ T cells were co-cultured with MDSC with or without E2, ICI 182,780, and JSI-124 for 48 hours. T-cell proliferation was evaluated by flow cytometry. CFSE was purchased from Tonbo Biosciences (San Diego, CA, USA).

For the proliferation assay with 1.5 µm carboxyfluorescein succinimidyl ester (CFSE) (#150347‐59‐4; Dojindo, Rockville, MD, USA), 293/null cells were resuspended in growth medium at a density of 10⁶·mL⁻¹ and cultured in six‐well plates with 0, 10 or 20 µm fatostatin. CFSE‐treated cells that were cultivated in the absence of DMSO or fatostatin were used as negative controls. Cells were labeled with CFSE in accordance with the manufacturer’s instructions (#13‐0850‐U50; TONBO Biosciences, San Diego, CA, USA). The 293/null cells were incubated at 37 °C in 5% CO₂ and harvested 44 h after fatostatin treatment. Cells were washed once with 1 × phosphate‐buffered saline and 1 × binding buffer (#00‐0055; Invitrogen, Waltham, MA, USA) each, at 620 g for 3 min. The pellet was resuspended in 200 µL of 1 × binding buffer, and incubated on ice for 15 min after the addition of 5 µL of 7‐AAD Viability Staining Solution (#559925; BD, Franklin Lakes, NJ, USA). Cell divisions were measured using a LSRFortessa cytometer (BD) and data were analyzed using flowjo, version 10.4 (BD).