Impact of washing-medium temperature on viability (A) and PBMC recovery (B).

<p>20 donors included. Ref.: reference group. **: p<0.01.</p

Gating strategy to identify live/dead CD45+ leukocytes.

<p>A) Events were triggered on FSC-H at a deliberately low threshold to avoid accidental exclusion of dead cells. B) Doublet exclusion C) Identification of CD45 positive cells D) Dead cells identified as 7-AAD positive events. Compensation for spectral overlap was not required.</p

Temperature of centrifuge by PBMC viability (A) and absolute live PBMC recovery (B).

<p>Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.</p

Comparing two ways of mixing PBMCs and washing medium by viability (A) and absolute count of live PBMCs (B).

<p>Paired samples 20 donors included. Ref.: reference group. NS: non-significant. **: p<0.01.</p

Centrifugation time and force and PBMC viability (A) and absolute live PBMC count (B).

<p>Paired samples from 18 donors included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.</p

PBMC viability (A) and absolute recovery (B) as a result of varying thawing time in 37°C heated water bath.

<p>Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.</p

PBMC viability (A) and absolute live PBMC recovery (B) by incubation.

<p>Samples were stored in a 37°C incubator with 5% CO₂. Paired samples from 20 donors were included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.</p

Granulocyte concentration in peripheral blood after CA4P treatment.

<p>A) and C) Granulocytes/ml peripheral blood as a function of hours after treatment with 25 mg/kg (• ○) or 250 mg/kg (▪ □) CA4P in C3H mammary carcinoma bearing (• ▪) and non-tumor bearing (○ □) CDF1 mice (n = 5–15). Blood samples from the saline-treated groups are depicted in the graphs at t = 0 h. In A) ▴ shows the granulocyte concentration in tumor bearing mice 1 h (depicted at t = 0) and 144 h after a saline injection (n = 15). Kruskal-Wallis One Way Analysis of Variance on Ranks was used to analyze for changes over time within each CA4P dose compared to t = 0 in each group. C) is a close up of the first 24 hours after treatment (shown in A). B) Granulocyte concentration as a function of tumor volume in CDF1 mice (n = 14). Pearson’s correlation coefficient = 0.88, p<0.001. D) Granulocytes/ml peripheral blood 1 h after treatment with PBS or 25 mg/kg CA4P in SCCVII squamous cell carcinoma bearing C3H/HeN mice (n = 10, Student’s t-test p<0.001). Data is presented as median values and the 25^th and 75^th percentiles (error bars) in A) and C), as single values in B), and as mean ± SEM in D).</p

The effect of CA4P treatment on cytokine levels in C3H mammary carcinomas in CDF1 mice.

<p>A) VEGF, B) MIP-1α (CCL3), C) KC (CXCL1), and D) MIP-2 (CXCL2) concentration in tumors as a function of hours after treatment with 25 mg/kg CA4P and in tumors from saline-treated mice. A) and C) Data is presented as mean ± SEM. B) and D) Data is presented as the median (horizontal bar), the 25^th and 75^th percentile (bottom and top of boxes) and the 10^th and 90^th percentiles (error bars). For all data n = 10.</p

Verification of neutrophil-depletion in CDF1 and C3H/HeN mice.

<p>A) Granulocytes (percentage of leucocytes) in non-tumor bearing CDF1 mice as a function of hours after treatment (n = 5). The mice were treated with PBS (○), the anti-neutrophil antibody 1A8 (•), CA4P (25 mg/kg, ▿), 1A8 and CA4P (25 mg/kg) in combination (▴), CA4P (250 mg/kg, □), or 1A8 and CA4P (250 mg/kg) in combination (▪). B) Granulocytes as a percentage of leucocytes in C3H/HeN mice 24 h after injection of PBS, 1A8, CA4P (25 mg/kg), or 1A8 and CA4P in combination (n = 5). The data is presented as median values and error bars represent the 25^th and 75^th percentiles.</p