anti-B220/CD45R (BD BioSciences; #553088;
1:100), and APC-conjugated anti-TCRb(BD
BioSciences; #553174; 1:100). Cells were stained
20 min on ice in the dark, after which 200ml of
DMEM was added and cells were analyzed via
a FACSCanto X (BD BioSciences). Cell counts
within 60 s were noted.
Transwell migration assay
To perform transwell migration assays using
peritoneal immune cells, 500mlofCMwas
added to a 24-well plate. A transwell inset
(3-mm pore size, Costar, #3415 or #3472) was
loaded with ~2×10^5 peritoneal immune cells
in 100ml of medium (matching the medium
used for CM production). Cells were allowed
to migrate for 12 hours. Then, the transwell
was carefully removed and the medium con-
taining suspension cells was collected. Attached
cells on the well bottom were washed twice
with PBS, trypsinized and scraped. Suspension
cells and attached cells were spun at 500gfor
10 min, resuspended and counted. Cell counts
were normalized to cell numbers of control
condition (CM cycling cells or CM EV) for each
mouse separately. For CXCL14 neutralization
experiments, CM from EV- or p21-OE cells was
added to a 24-well plate together with 20mg/ml
of goat anti-CXCL14 (R&D Systems, #AF866)
or 20mg/ml of goat anti-IgG (R&D Systems,
#AB-108-C) ( 73 ). Transwell migration assays
were performed as described above.
Injection of CM in wildtype mice
To determine the immune cell-eliciting po-
tential of CM, CM was generated as described
above except that culture medium with 0.5%
FBS was used. One milliliter of CM was aspi-
rated with a 25G needle and 3-ml syringe.
The needle was switched to 27G and CM was
slowly injected into the peritoneum of 8-
10-week-old C57BL/6 wildtype mice. Four days
post-injection, the peritoneal lavage was har-
vested and subjected to antibody staining and
flow cytometry as described above.
Statistical analysis
Prism software (GraphPad Software) was used
for statistical analyses. Unless otherwise stated,
Student’s two-tailed pairedttests (in MEFs and
HDFs) or Student’s two-tailed unpairedttests
(in IMR-90 cells and HUVECs) were used for
pairwise significance involving two groups. For
all experiments involving three or more groups,
one-way analysis of variance (ANOVA) with
Sidak’s correction or two-way ANOVA with
Sidak’s or Bonferroni correction for multiple
comparisons were performed. In these compar-
isons, the following denotes significance in all
figures:P<0.05,P<0.01, andP<0.001.
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