eBook: Breakthroughs and Innovations in Immunology Research - 14

576302, 10 ng/mL; Cat. No. 574302, 15 ng/mL).
Media was replenished every 2 days. After 7 days
in culture, cells were stimulated with LPS (50 µg/
mL) or CpG ODN 2395 (5 µM) for 12 to 16 hours.
After stimulation, the supernatant was collected
and kept at -20°C until processing for cytokine and
chemokine detection. Cells were recovered from
the plate and analyzed by flow cytometry.
Flow cytometry analysis
Cells were processed and stained following BioLegend's
protocols. Briefly, single cell suspensions
were prepared from bone marrow as described
previously, or lymphoid organs, and red cells were
lysed using Red Blood Cell Lysis Buffer (BioLegend
Cat. No. 420301, diluted 1 in 10). Cells were washed,
counted, and resuspended at the appropriate concentration
for staining. Non-specific binding was
blocked using TruStain FcX™ (anti-mouse CD16/32)
antibody (Cat. No. 101319). We now recommend
TruStain FcX™ PLUS (anti-mouse CD16/32) antibody
(Cat. No. 156603, clone S17011E) for higher
blocking capacity. Following Fc blocking, cells were
stained with anti-CD115 (Cat. No. 135505), CX3CR1
(Cat. No. 149007), CD80 (Cat. No. 104729), CD86
(Cat. No. 105013), I-A/I-E (MHC II) (Cat. No. 107627),
Ly-6G (Cat. No. 127625), Ly-6C (Cat. No. 128023),
and 7-AAD (Cat. No. 420403). Cells were acquired
with an LSR Fortessa™ (BD Biosciences) and analyzed
using FlowJo software.
Cytokine and chemokine measurement
Cytokine and chemokine profile was characterized
using BioLegend's LEGENDplex™ system. A detailed
protocol has been published.5
Briefly, LEGENDplex™
is a bead-based immunoassay that employs
the same basic principle as sandwich immunoassays.
Capture beads, which can be differentiated
by size and internal fluorescence intensity, are conjugated
to antibodies specific to a particular analyte.
A panel of defined capture beads is incubated
with the sample containing target analytes specific
to the capture antibodies. Then a biotinylated
detection antibody cocktail is added, which leads
to the formation of capture bead-analyte-detection
antibody sandwiches. Finally, streptavidinphycoerythrin
(PE) is added, which binds to biotinylated
detection antibodies, providing fluorescent
signal intensities in proportion to the amount
of bound analyte. The PE fluorescent signal of analyte-specific
bead regions is quantified using flow
cytometry, and the concentrations of particular analytes
are determined using data analysis software
and the standard curve generated in the assay.
In vivo cell transfer
CX3CR1+
cells were isolated as described above,
and labeled with CFSE following BioLegend's recommendations.
Recipient mice were anesthetized
with isoflurane and 106
cells were injected retro-orbitally
in 100 μL of PBS. Control animals received
100 μL of PBS only. 72 hours post-transfer, the mice
were sacrificed and spleen, bone marrow, and lymphoid
organs were collected and processed for
flow cytometry analysis. Cells were stained with
anti-CD115 (Cat. No. 135505), CX3CR1 (Cat. No.
149007), I-A/I-E (MHC II) (Cat. No. 107627), Ly-6C
(Cat. No. 128023), and 7-AAD (Cat. No. 420403).
Cells were acquired with an LSR Fortessa™ (BD Biosciences)
and analyzed using FlowJo software.
Results
Effective enrichment of bone marrow
CX3CR1+
cells using MojoSort
positive selection
The bone marrow is found within the central cavities
of axial and long bones. It consists of hematopoietic
tissue islands and adipose cells surrounded
by vascular networks embedded in trabecular
bone. It is the major hematopoietic organ as well as
14
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