Guava easy cyte 6ht 2l flow cytometer

Matrigel and the surrounding hind limb muscle were harvested from euthanized mice and enzymatically digested with Collagenase A (1 mg/mL; Roche Life Science) and Dispase (2.5 U/mL; BD Biosciences) for 2 h at 37 °C. The retrieved cells were incubated with PerCP-conjugated anti-mouse CD45 (1:100; BD Biosciences), PE-conjugated anti-mouse CD11b (1:100; BD Biosciences), PE-conjugated anti-mouse F4/80 (1:50; AbD Serotec), FITC-conjugated mLy6G (1:50; eBiosciences) and APC-conjugated Ly6C(1:100, eBioscience). Flow cytometric analyses were performed using a Guava easyCyte 6HT/2L flow cytometer (Millipore Corporation, Billerica, MA) and FlowJo software (Tree Star Inc., Ashland, OR). To determine ECFC and MPC retention, the retrieved cells were stained with PerCP-conjugated anti-mouse CD45, FITC-conjugated anti-human CD31 (1:10 BD Biosciences), and PE-conjugated anti-human CD90 (1:100 BD Biosciences). Mouse endothelial and stromal cell recruitment was stained by PerCP-conjugated anti-mouse CD45, PE-conjugated anti-mouse CD29 (1:100 eBioscience) and APC-conjugated anti-mouse CD31 (1:100 eBioscience).

Absolute number and viability of cells were determined with the ViaCount Assay performed on easyCyte 6HT-2L Guava flow cytometer (Millipore) following manufacturer's instructions. Red blood cells were lysed by transferring blood into a Tris-NH₄Cl 140mM, pH 7.5 solution and incubated at room temperature for 20 min. Cells were then centrifuged (300 g, 5 min, 20°C) and a second red blood cell lysis was performed if necessary. Cells were thereafter washed twice in phosphate buffered saline (PBS) containing 5% fetal bovine serum (FBS) and cell number and viability were again determined in PBLs with the ViaCount Assay. One to five millions of PBLs were processed in each staining combination.

Epidermis and hypodermis were carefully removed and tumor tissues were weighted. Single cell suspensions were obtained by an enzymatic treatment for 1 h at 37 °C under agitation with collagenase B at 4 mg/mL and DNase I at 0.1 mg/mL (Roche) in DMEM containing 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM l-Glutamine, 0.5 mM EDTA and 2% FBS (all from Gibco). Extensive washings were performed in the same medium without enzymes to remove released melanin.
Absolute number and viability of cells were determined with the ViaCount Assay performed on easyCyte 6HT-2L Guava flow cytometer (Millipore) following manufacturer’s instructions. Cells were stained as previously described for blood cell analysis [15 (link)]. Briefly, three combinations of antibodies, listed in supplementary Table 4, with the aqua LIVE/DEAD^® Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) for viability, were used to identify lymphoid cells (combinations A: CD45, CD3, CD8α, CD4, γδTCR and CD16 and B: CD45, MHC II, CD21 and CD79a) and myeloid cells (combination C: CD45, MHC II, PG68A, CD163, CD172a, CD14 and CADM1). Five million cells were processed in each combination and finally fixed in BD CellFIX solution before analysis on a BD LSR Fortessa cytometer (BD Biosciences). Data were analyzed using FlowJo V10 software (supplementary Table 5).

Cell suspensions were prepared by removing the epidermis and hypodermis, decontaminating the tissues for 30 min in solution A, 0.2% Fungizone (Gibco). Samples were weighed, minced, and enzymatically treated⁵² with 4 mg/mL collagenase B, 0.1 mg/mL DNase I (Roche) in DMEM containing 100 units/mL penicillin, 100 µg/mL streptomycin, 2mM L-glutamine, 0.5mM EDTA, and 2% FBS (all from Gibco) twice 20 min at 37 °C with shaking. Intensive washes were performed in the same medium without enzymes to remove released melanin. Absolute cell number and viability were determined using the ViaCount assay on an easyCyte 6HT-2L Guava flow cytometer (Millipore) according to the manufacturer’s instructions.

After the medium was removed, coculture cell layers were washed with PBS. Cells were detached from the plate using TrypLE Express. After blocking the unspecific binding sites with 100 μL 10% Gamunex (human immune globulin solution) for 10 min on ice, cells were incubated with the required antibodies (all antibodies were purchased from the Biolegend company) for further 30 min on dark. After centrifugation, cells were washed twice with FACS buffer (PBS containing 0.1% BSA) and measured by the Guava EasyCyte 6HT-2L flow cytometer (Merck Millipore, Germany) immediately. FlowJo software (FlowJo LLC, Ashland, OR, USA) was used for data evaluation.