Ultracomp beads ebeads

Flow cytometry was performed as previously described,32 (link) using fluorescent beads (UltraComp Beads eBeads, 176 Invitrogen, #01-2222-42) to determine compensation and fluorescence minus one methodology to determine gating (online supplemental figure S2). For in vivo analysis, tumors and tumor draining lymph nodes were harvested and gently dissociated. For in vitro analysis, nonadherent CD4 and CD8 cells were collected from culture plates and washed with PBS twice. B16 tumor cells or macrophages were washed with PBS and collected via gentle scraping. In either case total cells were treated CD16/32 antibody (BioLegend) to prevent non-specific binding. Live cell staining was performed using Ghost Red Dye 780 (Tonbo Biosciences) according to manufacturer’s instruction. After live-dead staining, a single cell suspension was labeled with the surface antibodies at 4°C for 60 min and washed three times using flow buffer (2% FBS+2 mM EDTA in PBS). For intracellular staining, the cells were fixed and stained for internal markers with permeabilization solution according to manufacturer’s instructions (BD Cytofix/CytopermTM). Flow cytometry was performed using an Attune NxT Flow Cytometer (ThermoFisher). Data were analyzed using FlowJo Software. Complete list of antibody targets, clones, and fluorophores is provided as online supplemental figure S2.

Tumor tissues were
collected at day 15 after the initiation of treatment from the B78
two-tumor bearing mice. The samples were enzymatically dissociated
with DNase and collagenase on a Gentle MACS Octodissociator (Miltenyi
Biotec) and then filtered through a 70 μm cell strainer and
red blood cells were lysed using RBC lysis buffer. The single cell
suspensions were divided for innate immune cell staining and adaptive
immune cells staining separately. For innate immune cell staining,
the cells were stained with GhostRed 780, followed by incubating with
anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained
with anti-CD45 PE-Cy7, anti-CD11b BV711, anti-F4/80 PE-Dazzle 594,
anti-CD80 PE, anti-CD206 APC, anti-CD11c FITC, and anti-MHCII BV510.
For adaptive immune cell staining, the cells were stained with GhostRed
780, followed by incubating with anti-CD16/CD32 (Fc block) for 5 min.
Then, the cells were stained with anti-CD45 PE-Cy7, anti-CD3 BV510,
anti-CD4 FITC, anti-CD8a Alexa 700, anti-NK1.1 PE-Cy5, anti-CD69 PerCP-Cy5.5,
and anti-CD25 APC. After the cells were fixed and permeabilized, they
were stained with anti-FOXP3 PE. Flow cytometry was performed on an
Attune Cytometer (ThermoFisher), and UltraComp Beads eBeads (Invitrogen)
were used for compensation. The flow cytometry data was analyzed using
FlowJo 10.6.2. The M1/M2 macrophage ratios were calculated with the
counts of the cells.

TDLNs were collected at day 15 after the initiation of treatment
from the B78 two-tumor bearing mice. The samples were enzymatically
dissociated with DNase and collagenase on a Gentle MACS Octodissociator
(Miltenyi Biotec) and then filtered through a 70 μm cell strainer.
The red blood cells were lysed using RBC lysis buffer. For innate
immune cell staining, the cells were stained with GhostRed 780, followed
by incubating with anti-CD16/CD32 (Fc block) for 5 min. Then, the
cells were stained with anti-CD45 PE-Cy7, anti-CD11b BV711, anti-F4/80
PE-Dazzle 594, anti-CD80 PE, anti-CD206 APC, anti-CD11c PerCP-Cy5.5,
and anti-MHCII BV510. For adaptive immune cell staining, the cells
were stained with GhostRed 780, followed by incubating with anti-CD16/CD32
(Fc block) for 5 min. Then, the cells were stained with anti-CD45
PE-Cy7, anti-CD3 BV510, anti-CD4 FITC, anti-CD8a Alexa 700, anti-CD69
BV421, anti-CD44 PE, and anti-CD62L PE-Cy5. Flow cytometry was performed
on an Attune cytometer (ThermoFisher), and UltraComp Beads eBeads
(Invitrogen) were used for compensation. The flow cytometry data was
analyzed using FlowJo 10.6.2.

Blood was collected
at day 15 after the initiation of treatment from the B78 two-tumor
bearing mice. The red blood cells were lysed using RBC lysis buffer.
The cells were stained with GhostRed 780, followed by incubating with
anti-CD16/CD32 (Fc block) for 5 min. Then, the cells were stained
with anti-CD45 PE-Cy7, anti-CD3 BV510, anti-CD4 FITC, anti-CD8a Alexa
700, anti-PD-1 BV421, anti-CD44 PE, and anti-NKG2D PE-Dazzle 594.
Flow cytometry was performed on an Attune cytometer (ThermoFisher),
and UltraComp Beads eBeads (Invitrogen) were used for compensation.
The flow cytometry data was analyzed using FlowJo 10.6.2.