Moflo astrios eq high speed cell sorter

MDA-MB-231-eGFP-mCherry cells were washed twice with warm washing buffer (1× PBS + 10 mM EDTA) followed by short incubation with 3 mM EDTA in 1× PBS. Semi-detached cells were treated with TrypLE followed by dilution in 1× PBS + 10 mM EDTA. Cells were centrifuged at 350×g for 5 min, washed with 1× PBS + 10 mM EDTA and resuspended in sorting buffer (ice cold 1× PBS, 5 mM EDTA, 1% FCS and 25 mM HEPES; pH 7.0). Cells were then filtered using a 30 μm Filcon sterile filter (BD Biosciences) and sorted on the basis of fluorescence intensity compared to control cells including parental MDA-MB-231 (no colour), MDA-MB-231-eGFP (single colour) and MDA-MB-231-mCherry (single colour). Single cells separated in 96-well plates were grown in the conditioned media before transferring into larger 6-well plates. Flow Cytometry sorting was performed on the MoFlo Astrios EQ High Speed Cell Sorter using Summit Software version: 6.3.1 (Beckman Coulter, Miami, FL, USA). Experiments utilised the 488 nm (150 mW) and 561 (200 mW) laser lines and the 100 micron nozzle at 30 PSI. Laser and light filter usage are displayed on plot axes. No forward scatter masks were used. Flow cytometry data was analysed using the Apple Macintosh-version of FCS express 6 (De Novo Software, Los Angeles, CA, USA).

Bovine cells were suspended in PBS with 10% of horse serum (Gibco), 2mM EDTA, and labeled for 30 min with primary antibodies (see Supplementary Table S1). Mouse cells were suspended in PBS 2mM EDTA. After saturation with anti-CD32/CD16, murine cells were incubated 30min with fluorescent mAb. After washes in D-PBS (300xg, 10 min, 4°C), both murine and bovine cells were labeled 30min with the corresponding fluorescent-conjugated secondary antibodies. Cells were washed and fixed with BD cell fix diluted four times in PBS. Data were acquired with a LSR Fortessa™ X-20 Flow cytometer (Becton Dickinson) and results analyzed with Kaluza software (Beckman Coulter).
For neutrophil subsets purification, cell concentrations were adjusted to 10⁷cells/ml and sorted with a MoFlo Astrios^EQ high speed cell sorter (Beckman Coulter) according to our previously published protocol (18 (link)). Sorted cells were spread on microscope slides (Superfrost, Thermo) by cytocentrifugation (3 min, 700xg) and stained with May-Grünwald and Giemsa with the RAL 555 kit (RAL Diagnostics).

The testes at P2 were collected, de‐capsulated and digested with TrypLE™ Express Enzyme (Thermo Fisher, 12605010). The cell suspensions were filtered through a 40 μm cell strainer (Corning, 22363547), and cells were collected by centrifugation at 300 × g for 5 minutes. The pellets were resuspended in DPBS (HyClone, SH30028) with 0.04% BSA and isolated with a MoFlo Astrios EQ high speed cell sorter (Beckman Coulter). Then, the cells were sorted into cell lysis buffer containing 0.45% (vol/vol) NP40 (Roche, 11332473001), followed by reverse transcription using SuperScript II Reverse Transcriptase (Invitrogen, 18064‐014) and whole transcription amplification using KAPA HiFi HotStart Ready Mix (KAPA Biosystems, KK2602).²⁷