Amnis imaging flow cytometer

Reticulocytes were sorted based on CD235a and CD71 expression by the AriaIII Cell sorter (BD Biosciences, Franklin Lakes, USA) lysed in Laemmli sample buffer and subjected to SDS-PAGE (Whatman, Little Chalfont, UK). Erythroblasts and reticulocytes of mixed stages were cultured as described perviously^{14 (link)–16 (link)}. Imaging flow cytometry was performed on the Amnis Imaging Flow Cytometer (Millipore, Burlington, USA).

CIA-FLS were stimulated for 72 h with 100 ng/ml IFN-γ and loaded with 10 μg/ml ovalbumin for 2–3 h in the presence of IFN-γ. The CIA-FLS were then labeled with CellTrace Violet dye (Thermo Fisher Scientific, Waltham, MA, USA), and ovalbumin-specific T_EM cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher Scientific). The CIA-FLS were then removed from their culture flasks and placed in 1.5-ml Eppendorf tubes with the ovalbumin-specific T_EM cells at a 4:1 ratio of T_EM cells to CIA-FLS. The cocultures were briefly centrifuged, incubated at 37 °C for 30 min in the presence or absence of either 20 μM paxilline or 100 nM ShK-186, and fixed with 4% paraformaldehyde. Cells were then stained with an anti-CD3 antibody conjugated to APC (antibody clone 1F4; BD Biosciences) and analyzed with an Amnis Imaging Flow Cytometer (MilliporeSigma) to image cell conjugates and the formation of an immune synapse or with a FACSCanto II flow cytometer to quantify cell conjugates, with gating completed to exclude single cells.

Blood samples were collected from patients at baseline. In addition, tumor tissues obtained from patients were prepared as single cell suspension as described below. All samples were immunostained with various antibodies to identify specific cell populations. Specifically, white blood cells were defined as CD45+. Of those, early myeloid cells were defined as CD33+HLA-DR-. Of these, further division was made based on the presence or absence of CD11b/CD16 surface markers. The antibody mix included anti HLA-DR-FITC, anti-CD33-PE, anti CD11b-APC, anti-CD45-APC-Cy7, anti-CD16-PerCP, anti-CD3-PE, and anti-CD8-FITC. All antibodies were purchased from BioLegend. For whole blood samples, after immunostaining, the samples underwent red blood cell lysis. All samples were analyzed by Amnis® imaging flow cytometer using IDEAS® software (Millipore Sigma).

Single-cell suspensions from brains or PBMCs were stained with an antibody cocktail. The following antibodies were used: anti-CD3–FITC (11-0031, Thermo Fisher Scientific; 1:400), anti-CD8–eFluor 450 (48-0081, Thermo Fisher Scientific; 1:400), anti-CD122–PECy7 (25-1222, Thermo Fisher Scientific; 1:400), anti-CXCR3–BV510 (745033, BD Biosciences; 1:200), and rabbit anti–mouse ETGF (ab9585, Abcam; 1:400) subsequently bound with anti-rabbit–Alexa Fluor 488. Fluorochrome compensation was performed with single-stained OneComp eBeads (01-1111-41, Thermo Fisher Scientific). ImageStream analysis was performed using an Amnis Imaging Flow Cytometer (MilliporeSigma). Data analysis was performed using IDEAS software (MilliporeSigma).

Cells were washed in permeabilization buffer (eBioscience, Thermo Fisher Scientific) and stained overnight at 4°C with FITC-conjugated mouse anti γ-H2AX (Serine139) (1:2000, MilliporeSigma, 16-202A) and Alexa Fluor 647–conjugated rabbit anti-53BP1 (1:400, Novusbio, NB100-304). This was followed by staining with DAPI (1:5000) (Thermo Fisher Scientific) for 5 minutes. Cells were then analyzed with AMNIS imaging flow cytometer (MilliporeSigma). At least 1000 cells were captured per condition at original magnification, ×40, with extended depth of field. Data were analyzed using IDEAS 6.2 imaging flow cytometry software (MilliporeSigma) (23 (link)). Full details for the gating strategy are described in Supplemental Methods.