Surface staining of either erylysed blood
or splenocyte suspension (for sorting for T cells and B cells) or
lineage-depleted cells (for sorting stem/progenitor cells) was performed
by resuspending the cell pellet in ice-cold PBS containing the surface
antibody cocktail A (CD4-PeCy5 (Cat# 100410, 1:1000), CD8a-PeCy5 (Cat#
100710, 1:2000), B220-AF700 (Cat# 103231, 1:300)) for sorting B and
T cells from blood, antibody cocktail B (CD4 (Cat# 100422, 1:1000)/CD8a
(Cat# 100722, 1:1000)/B220 (Cat# 103222, 1:1000)/Ter119 (Cat# 116221,
1:500)/Gr-1 (Cat# 108416, 1:500)/CD11b (Cat# 101216, 1:1000)-all PeCy7,
c-Kit/CD117-PE (Cat# 105808, 1:1000), Sca-1-APC-Cy7 (Cat# 108126,
1:500), CD150-BV605 (Cat# 115927, 1:200), CD48-BV421 (Cat# 103428,
1:500)) for sorting stem/progenitor cells, or antibody cocktail C
(CD3-PE (Invitrogen Cat# 12-0031-83, 1:200), B220-A647 (Cat# 103226,
1:200)) for sorting B and T cells from spleen, followed by incubation
for 30 min at 4 °C in the dark. All antibodies were purchased
from BioLegend unless noted otherwise. Mast cells and macrophages
were stained with the Zombie NIRTM fixable viability dye (BioLegend,
Cat# 423105; 1:200) in 500 μL of Opti-MEM per sample for 10
min at 20 °C in the dark.
Cells were washed with 1 mL of
ice-cold PBS and centrifuged at 400g for 5 min at
4 °C, and the supernatant was then discarded. Depending on the
size of the pellet, the cells were resuspended in an appropriate volume
of ice-cold physiological water (9 g/L NaCl, usually between 500 μL
and 2 mL) containing a 1:200 ratio internal standard stock. The internal
standard stock was 6 mg/mL chloro-phenylalanine and 6 mg/mL aminoterephthalic
acid in 30% v/v 2-propanol in water. Finally, cells were filtered
through a 30 μm cell strainer (mast cells and macrophages) or
filter-cap FACS tube (all other cell types) to immediately proceed
with flow cytometry-based sorting.
or splenocyte suspension (for sorting for T cells and B cells) or
lineage-depleted cells (for sorting stem/progenitor cells) was performed
by resuspending the cell pellet in ice-cold PBS containing the surface
antibody cocktail A (CD4-PeCy5 (Cat# 100410, 1:1000), CD8a-PeCy5 (Cat#
100710, 1:2000), B220-AF700 (Cat# 103231, 1:300)) for sorting B and
T cells from blood, antibody cocktail B (CD4 (Cat# 100422, 1:1000)/CD8a
(Cat# 100722, 1:1000)/B220 (Cat# 103222, 1:1000)/Ter119 (Cat# 116221,
1:500)/Gr-1 (Cat# 108416, 1:500)/CD11b (Cat# 101216, 1:1000)-all PeCy7,
c-Kit/CD117-PE (Cat# 105808, 1:1000), Sca-1-APC-Cy7 (Cat# 108126,
1:500), CD150-BV605 (Cat# 115927, 1:200), CD48-BV421 (Cat# 103428,
1:500)) for sorting stem/progenitor cells, or antibody cocktail C
(CD3-PE (Invitrogen Cat# 12-0031-83, 1:200), B220-A647 (Cat# 103226,
1:200)) for sorting B and T cells from spleen, followed by incubation
for 30 min at 4 °C in the dark. All antibodies were purchased
from BioLegend unless noted otherwise. Mast cells and macrophages
were stained with the Zombie NIRTM fixable viability dye (BioLegend,
Cat# 423105; 1:200) in 500 μL of Opti-MEM per sample for 10
min at 20 °C in the dark.
Cells were washed with 1 mL of
ice-cold PBS and centrifuged at 400g for 5 min at
4 °C, and the supernatant was then discarded. Depending on the
size of the pellet, the cells were resuspended in an appropriate volume
of ice-cold physiological water (9 g/L NaCl, usually between 500 μL
and 2 mL) containing a 1:200 ratio internal standard stock. The internal
standard stock was 6 mg/mL chloro-phenylalanine and 6 mg/mL aminoterephthalic
acid in 30% v/v 2-propanol in water. Finally, cells were filtered
through a 30 μm cell strainer (mast cells and macrophages) or
filter-cap FACS tube (all other cell types) to immediately proceed
with flow cytometry-based sorting.
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