can be acquired only during the initial optimization
experiment.
17. Fix cells by adding either 100 or 250 μL of Cytofix™ Fixation
Buffer per well or tube, respectively. Mix thoroughly by vortexing, and incubate for 15–30 min at 4
C in the dark.
Perform the same wash steps as described previously, and
resuspend in 500 μL staining buffer prior to sample acquisition. Fixed cells can be stored at 4
C in the dark for up to
a week.
18. Flow cytometry data from our immunization studies are routinely analyzed using FlowJo™ 10 (BD). The total number of
each immune cell type is determined using the formula: (percentage of immune cell type gated on live CD45
+ cells  total
sample cell number obtained using the Cellometer Auto 2000
cell viability counter)/100. Similarly, the number of
OVA-AF647
+ cells per immune cell type is calculated using
the formula: (percentage of OVA-AF647
+ immune cell type
gated on live CD45
+ cells  total sample cell number obtained
using the Cellometer Auto 2000 cell viability counter)/100.
Immune cell numbers are graphed and analyzed for statistical
significance using GraphPad
® Prism 8 (GraphPad, La Jolla,
CA, USA).
Acknowledgments
The authors would like to acknowledge Mario Mercier and Shawn
Makinen for their technical assistance with the i.m. injections and
tissue collections.
References
1. Haq K, Jia Y, Krishnan L (2016) Archaeal lipid
vaccine adjuvants for induction of cellmediated immunity. Expert Rev Vaccines
15:1557–1566
2. Akache B, Stark FC, Jia Y, Deschatelets L,
Dudani R, Harrison BA, Agbayani G,
Williams D, Jamshidi MP, Krishnan L et al
(2018) Sulfated archaeol glycolipids: comparison with other immunological adjuvants in
mice. PLoS One 13:e0208067
3. Akache B, Stark FC, Iqbal U, Chen W, Jia Y,
Krishnan L, McCluskie MJ (2018) Safety and
biodistribution of sulfated archaeal glycolipid
archaeosomes as vaccine adjuvants. Hum Vaccin Immunother 14:1746–1759
4. Jia Y, Akache B, Deschatelets L, Qian H,
Dudani R, Harrison BA, Stark FC,
Chandan V, Jamshidi MP, Krishnan L et al
(2019) A comparison of the immune responses
induced by antigens in three different
archaeosome-based vaccine formulations. Int
J Pharm 561:187–196
5. McCluskie MJ, Deschatelets L, Krishnan L
(2017) Sulfated archaeal glycolipid archaeosomes as a safe and effective vaccine adjuvant
for induction of cell-mediated immunity. Hum
Vaccin Immunother 13:2772–2779
6. Anesthesia Induction and Maintenance. 2019.
JoVE, Cambridge, MA, JoVE science education database. Lab Animal Research. Ref Type:
Audiovisual Material
Cell Recruitment and Antigen Uptake and Distribution
523
experiment.
17. Fix cells by adding either 100 or 250 μL of Cytofix™ Fixation
Buffer per well or tube, respectively. Mix thoroughly by vortexing, and incubate for 15–30 min at 4
C in the dark.
Perform the same wash steps as described previously, and
resuspend in 500 μL staining buffer prior to sample acquisition. Fixed cells can be stored at 4
C in the dark for up to
a week.
18. Flow cytometry data from our immunization studies are routinely analyzed using FlowJo™ 10 (BD). The total number of
each immune cell type is determined using the formula: (percentage of immune cell type gated on live CD45
+ cells  total
sample cell number obtained using the Cellometer Auto 2000
cell viability counter)/100. Similarly, the number of
OVA-AF647
+ cells per immune cell type is calculated using
the formula: (percentage of OVA-AF647
+ immune cell type
gated on live CD45
+ cells  total sample cell number obtained
using the Cellometer Auto 2000 cell viability counter)/100.
Immune cell numbers are graphed and analyzed for statistical
significance using GraphPad
® Prism 8 (GraphPad, La Jolla,
CA, USA).
Acknowledgments
The authors would like to acknowledge Mario Mercier and Shawn
Makinen for their technical assistance with the i.m. injections and
tissue collections.
References
1. Haq K, Jia Y, Krishnan L (2016) Archaeal lipid
vaccine adjuvants for induction of cellmediated immunity. Expert Rev Vaccines
15:1557–1566
2. Akache B, Stark FC, Jia Y, Deschatelets L,
Dudani R, Harrison BA, Agbayani G,
Williams D, Jamshidi MP, Krishnan L et al
(2018) Sulfated archaeol glycolipids: comparison with other immunological adjuvants in
mice. PLoS One 13:e0208067
3. Akache B, Stark FC, Iqbal U, Chen W, Jia Y,
Krishnan L, McCluskie MJ (2018) Safety and
biodistribution of sulfated archaeal glycolipid
archaeosomes as vaccine adjuvants. Hum Vaccin Immunother 14:1746–1759
4. Jia Y, Akache B, Deschatelets L, Qian H,
Dudani R, Harrison BA, Stark FC,
Chandan V, Jamshidi MP, Krishnan L et al
(2019) A comparison of the immune responses
induced by antigens in three different
archaeosome-based vaccine formulations. Int
J Pharm 561:187–196
5. McCluskie MJ, Deschatelets L, Krishnan L
(2017) Sulfated archaeal glycolipid archaeosomes as a safe and effective vaccine adjuvant
for induction of cell-mediated immunity. Hum
Vaccin Immunother 13:2772–2779
6. Anesthesia Induction and Maintenance. 2019.
JoVE, Cambridge, MA, JoVE science education database. Lab Animal Research. Ref Type:
Audiovisual Material
Cell Recruitment and Antigen Uptake and Distribution
523