centrifuge tube, and place in a shaking incubator for 1 h at
37
C and 100 rpm.
7. Inactivate Collagenase Type IV digestion buffer activity by
adding 10 mL of ice-cold R10 complete medium.
8. Centrifuge cells at 500 Â g for 5 min at 4
C. Remove supernatant and resuspend cells in 1 mL R10 complete medium.
9. Pass cells through a 100-μm nylon cell strainer placed onto a
50-mL Falcon™ conical centrifuge tube prior to cell counting.
3.4 Lymph Node
Collection
and Processing
1. Isolate the inguinal and popliteal LNs using surgical scissors
and forceps. Keep LNs in 1.5-mL microcentrifuge tubes containing 1 mL R10 complete medium on ice or at 4
C until
ready for tissue processing.
2. Place the LNs in 1 mL R10 complete medium onto a sterile
petri dish and homogenize using the rough edge of sterilized
frosted microscope slides.
3. Pass cells through a 100-μm nylon cell strainer placed onto a
50-mL Falcon™ conical centrifuge tube prior to cell counting.
3.5 Cell Counting
1. Dilute 15 μL of sample with 15 μL AO/PI viability stain (1:1
ratio).
2. Load 20 μL of sample stained with AO/PI viability stain onto a
counting chamber and analyze on the Cellometer Auto 2000
Cell Viability Counter (see Note 10).
3.6 Flow Cytometry
Staining
and Acquisition
1. Prepare single-stain controls using the UltraComp eBeads™
compensation beads (see Note 11).
2. Reconstitute a fresh vial of LIVE/DEAD™ Fixable Dead Cell
Stain by adding 50 μL of DMSO included in the kit.
3. Prepare a 1:100 working concentration of LIVE/DEAD™
Fixable Dead Cell Stain using 1Â PBS. A total of 50 μL working concentration is required per sample (see Note 12).
4. Transfer up to 1 Â 10
6 cells to a 5-mL polystyrene tube or to
each well of a 96-well DeepWell™ polypropylene microplate
(see Note 13).
5. Centrifuge cells at 500 Â g for 5 min at 4
C and remove
supernatant (see Note 14).
6. Add 50 μL of the working concentration (1:100) of LIVE/
DEAD™ Fixable Dead Cell Stain to each well or tube, and
incubate for 20 min at 4
C in the dark.
7. Wash cells by adding 1 mL 1Â PBS and centrifuging at 500 Â g
for 5 min at 4
C.
8. Remove supernatant and resuspend cells in 50 μL Fc block.
Incubate for 10 min at 4
C in the dark.
518
Gerard Agbayani et al.
37
C and 100 rpm.
7. Inactivate Collagenase Type IV digestion buffer activity by
adding 10 mL of ice-cold R10 complete medium.
8. Centrifuge cells at 500 Â g for 5 min at 4
C. Remove supernatant and resuspend cells in 1 mL R10 complete medium.
9. Pass cells through a 100-μm nylon cell strainer placed onto a
50-mL Falcon™ conical centrifuge tube prior to cell counting.
3.4 Lymph Node
Collection
and Processing
1. Isolate the inguinal and popliteal LNs using surgical scissors
and forceps. Keep LNs in 1.5-mL microcentrifuge tubes containing 1 mL R10 complete medium on ice or at 4
C until
ready for tissue processing.
2. Place the LNs in 1 mL R10 complete medium onto a sterile
petri dish and homogenize using the rough edge of sterilized
frosted microscope slides.
3. Pass cells through a 100-μm nylon cell strainer placed onto a
50-mL Falcon™ conical centrifuge tube prior to cell counting.
3.5 Cell Counting
1. Dilute 15 μL of sample with 15 μL AO/PI viability stain (1:1
ratio).
2. Load 20 μL of sample stained with AO/PI viability stain onto a
counting chamber and analyze on the Cellometer Auto 2000
Cell Viability Counter (see Note 10).
3.6 Flow Cytometry
Staining
and Acquisition
1. Prepare single-stain controls using the UltraComp eBeads™
compensation beads (see Note 11).
2. Reconstitute a fresh vial of LIVE/DEAD™ Fixable Dead Cell
Stain by adding 50 μL of DMSO included in the kit.
3. Prepare a 1:100 working concentration of LIVE/DEAD™
Fixable Dead Cell Stain using 1Â PBS. A total of 50 μL working concentration is required per sample (see Note 12).
4. Transfer up to 1 Â 10
6 cells to a 5-mL polystyrene tube or to
each well of a 96-well DeepWell™ polypropylene microplate
(see Note 13).
5. Centrifuge cells at 500 Â g for 5 min at 4
C and remove
supernatant (see Note 14).
6. Add 50 μL of the working concentration (1:100) of LIVE/
DEAD™ Fixable Dead Cell Stain to each well or tube, and
incubate for 20 min at 4
C in the dark.
7. Wash cells by adding 1 mL 1Â PBS and centrifuging at 500 Â g
for 5 min at 4
C.
8. Remove supernatant and resuspend cells in 50 μL Fc block.
Incubate for 10 min at 4
C in the dark.
518
Gerard Agbayani et al.