Flow cytometry and cell sorting

We blocked single-cell suspensions with a FcR blocking reagent (Miltenyi Biotec) in 0.5% PBS–BSA for 20 min, stained them with fluorochrome-conjugated antibodies, and analyzed them on a LSR II/Fortessa flow cytometer or sorted them using a FACS Aria III cell sorter (both from BD Biosciences). We analyzed data using FlowJo V10 (TreeStar). All antibodies and secondary reagents were titrated to determine optimal concentrations. Comp-Beads (BD Biosciences) were used for single-color compensation to create multicolor compensation matrices. For gating, we used fluorescence minus one controls. We controlled instrument calibration daily using Cytometer Setup and Tracking beads (BD Biosciences). For characterization and sorting of immune cell subsets in mouse tumors, we used the following antibodies: anti-CD3-PE-CF594, anti-CD4-BV510, anti-CD8-BV650, anti-CD11b-BV605, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326-BV711, anti-Ly6C-Per-CP-Cy5.5 (BD Biosciences), anti-CD45-Vio-Blu, anti-MHC-II-APC (Miltenyi Biotec), anti-CD80-PE, anti-F4/80-PE-Cy7, anti-CD206-FITC, and anti-Ly6G-APC-Cy7 (BioLegend). 

Detailed protocol:

FACS staining 

o   resuspend single cell pellet in 100 µl PBS/BSA (0.5%) and transfer to FACS tube 

•   Add 2µl FcR-block to samples

•   Incubate for 20 min at 4°C

•   Prepare antibody Master Mix (see below). Antibodies should be added into 50 µl brilliant violet buffer

•   Add Master Mix to samples and incubate for 10 min at 4°C in the dark

•   Add 2 µl 7AAD to samples and incubate for another 10 min at 4°C in the dark

•   Add 300 µl FACS Flow to samples and centrifuge for 5 min at 4°C at 500 x g

•   Remove supernatant completely

•   Resuspend pellet in 500 µl PBS and filter cell suspension through Cell-Strainer Cap of Round-Bottom Tube

•   Start analysis in LSR Fortessa 

•   Acquire FMO controls routinely for gating in FlowJo 

FACS panel mouse (antibody details in Han et al 2019 Cell Rep, PMID 30995480)

For the characterization and sorting of human macrophages, single-cell suspensions were stained with the following antibodies: anti-CD33-BV510, anti-CD45-AlexaFluor700, anti-CD64-BV605, anti-CD83-BV711 (BD Biosciences), anti-CD163-PE, anti-CD206-PE-Cy7, and anti-CD326-FITC (BioLegend). We used 7-AAD to exclude dead cells. 

Detailed protocol:

Generation of single cell suspensions. 

To dissociate tumor sample into a single cell suspension the Tumor Dissociation Kit (human) by Miltenyi Biotech was used. 

•   Prepare tumor digestion mix in Gentle MACS C-tube using reagents from Tumor Dissociation kit. Mix following reagents for one reaction: 

o   4.7 ml RPMI Medium (+10 µg/ml CHX)

o   200 µl of Enzyme H

o   100 µl of Enzyme R

o   25 µl of Enzyme A

•   Weight tumor tissue and write down weight

•   If tumor weight is above 1 g, divide sample in two tubes

•   Cut tumor in small pieces (2-4 mm) with scalpel and transfer into Gentle MACS C-tube

•   Connect tube to Gentle MACS Dissociator and run program “h_tumor_01”

•   Quick spin tube to collect sample at the bottom

•   Incubate sample for 20 min at 37°C and 130RPM in Incubator Innova®44

•   Reattach tube to Gentle MACS Dissociator and run program “h_tumor_01”

•   Quick-spin tube to collect sample at the bottom

•   Add 20 ml RPMI Medium (+10 µg/ml CHX) to sample and filter cell suspension trough 70 µm nylon mesh into 50 ml falcon

•   Spin down for 5 min, 500 x g, 4°C

•   Remove supernatant completely

•   Start FACS staining immediately

FACS staining 

o   resuspend pellet in 100 µl PBS/BSA (0.5%) and transfer to FACS tube 

•   Add 2µl Fc-block to samples

•   Incubate for 20 min at 4°C

•   Prepare antibody Master Mix (see below). Antibodies should be added into 50 µl brilliant violet buffer

•   Add Master Mix to samples and incubate for 10 min at 4°C in the dark

•   Add 2 µl 7AAD to samples and incubate for another 10 min at 4°C in the dark

•   Add 300 µl FACS Flow to samples and centrifuge for 5 min at 4°C at 500 x g

•   Remove supernatant completely

•   Resuspend pellet in 500 µl PBS and filter cell suspension through Cell-Strainer Cap of Round-Bottom Tube

•   Start analysis and/or sorting of myeloid populations

FACS-Sorting of myeloid populations with FACS Aria III

•   Prepare 6 x 1.5 ml DNA LoBind Tubes containing 1.0 ml PBS and invert them multiple times

•   Prepare 1 x 1.5 ml DNA LoBind Tube containing 500 µl sorting buffer (1x PBS, 3 mM EDTA, 25 mM HEPES, 2% FCS)

•   Load sample tube into sorter, acquire and slightly adjust gates if necessary 

•   Sort myeloid populations using an 85 µm nozzle  

•   Use them immediately for downstream applications

FACS panel human

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