40 um cell strainer

This assay was performed as described elsewhere [17 (link)], with minor modifications. Briefly, to prepare target cells, splenocytes from naive C57BL6 mice were harvested and red blood cells lysed. Half of the cells were loaded with influenza SA/wt virus at 0.5 MOI for one hour in complete RPMI-1640 medium without fetal bovine serum (FBS) at 37°C. The remaining cells were mock-loaded with diluted normal chicken egg allantoic fluid in culture medium. After incubation, mock- and virus-loaded cells were washed and stained with 20 mM and 2 mM of carboxyfluorescein succinimidyl ester (CFSE), respectively. Next, target cells were washed with Hanks balanced salt solution and filtered thought 40 um cell strainer (BD Bioscience, USA). Then mock- and virus-loaded target cells were mixed in a 1 : 1 ratio. 1.5 × 10⁶ target cells were administered in 100 μl to anesthetized mice on day 42 after the primary immunization, by retroorbital injection. Next day, the mice were sacrificed and splenocytes were harvested and processed by flow cytometry. Cytotoxicity was presented as virus- to mock-loaded cell count ratio.
Statistical analysis of the results was carried out using GraphPad Prizm 6 and Statistica 10 software. The nonparametric Mann–Whitney Test and Kruskal-Wallis test were applied for data comparison. p value < 0.05 was considered to be statistically significant.

For DNA content analysis, hPSCs cells were dissociated by TrypLE Express (Invitrogen) and fixed with 70% EtOH. Propidium iodide was used to for H1 and HUES8 cells and Hoechst 33342 (both Sigma) for FUCCI-H9 cells for 30 min at 37 ^oC to stain DNA. Samples were then fixed with 2% Paraformaldehyde/PBS (PFA), washed and filtered through 40 um cell strainer (BD Biosciences) before flow cytometry. FACS analysis was performed using LSRII (BD Biosciences) and analyzed using FACSDiva software. Gates were set with reference to negative controls.

Endometrial samples were digested for 60 min at 37 °C with 5 mg/mL of collagenase type I (Worthington, USA), 4 mg/mL of DNase 1 (Worthington), and 500 mM glucose (Sinopharm, China), with intermittent trituration every 20 min. The cell suspension was passed through a 40um cell strainer (BD Biosciences), washed in bench medium comprising 5% newborn calf serum (Sigma-Aldrich), 1% antibiotic/ antimycotic (Gibco) in DMEM/F12 (Gibco) before centrifuging over Ficoll^® Paque Plus (GE Healthcare) at 460 g, 15 min, 20 °C with brakes off. The mononuclear cells were collected, washed with bench medium before suspension in 2% fetal bovine serum (BSA, Sigma-Aldrich), in PBS and counted.

Antibodies with specificity for CD4, FoxP3, CD 206 and F4/80 were obtained from eBioscience. For cell number quantification, PE, PerCP and APC labeled counting beads (Invitrogen) were used. Explanted lungs were digested in collagenase containing PBS for 60 minutes at 37C. Single cell suspension was obtained by passing the cells through 40 um cell strainer (BD). For intracellular cytokine staining, cells were stained for CD4, fixed, and permeabilized using the Fix&Perm kit (BD) and stained with antibody to FoxP3. Cells were analyzed on a 4-color FACS Calibur instrument (BD) with FlowJo version 8.8.