host but it is also compared to the proportions of cells detected
after injection into naı ¨ve animals. Variation in Target–Nontarget cell ratios such as 55:45 (or similar) are normal; it is not
necessary to discard “Target” cells to ensure that an exact equal
number of cells are injected. The most important criteria is to
maintain a consistency in the number of “Target” cells injected
within an experiment or preferably as a standard for all
experiments.
17. Cells will be injected at a concentration of 100 million cells/ml
(20 million cells/200 μl/mouse). “Target” cells and “Nontarget” cells are counted separately to take note of any extreme
differences in cell count. Any extreme loss of cells (>50%)
could be a result of CFSE staining toxicity and more care
would be taken during these steps.
18. Typically, mice are euthanized 24 h after cell injection; however, for logistical purposes, we euthanize at 20 h and still
observe the same extent of target cell killing.
19. If samples cannot be acquired immediately, cover the deep-well
plate at this step and refrigerate until ready (up to 4 h). Since
live splenocytes can readily reform clumps, it is best to filter the
cells immediately before acquisition by flow cytometry.
20. If a plate reader is not available or if there are only a few
samples, cells can be individually transferred to 5 ml roundbottom polystyrene tubes and acquired manually.
21. Flow cytometry data from our immunization studies are routinely analyzed using FlowJo™ 10 (BD Biosciences, Franklin
Lakes, NJ, USA). CFSE
+ cells are graphed and analyzed for
statistical significance using GraphPad
® Prism 8 (GraphPad, La
Jolla, CA, USA).
References
1. Hollingsworth RE, Jansen K (2019) Turning
the corner on therapeutic cancer vaccines. NPJ
Vaccines 4:7
2. Garc ¸on N, Di Pasquale A (2016) From discovery to licensure, the adjuvant system story. Hum
Vaccin Immunother 13:19–33
3. De Gregorio E, Caproni E, Ulmer JB (2013)
Vaccine adjuvants: mode of action. Front Immunol 4:214
4. Landi A, Law J, Hockman D et al (2017) Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP,
a STING activator or archaeosomes. Vaccine
35:6949–6956
5. Wooldridge L, Lissina A, Cole DK et al (2009)
Tricks with tetramers: how to get the most from
multimeric
peptide–MHC.
Immunology
126:147–164
6. Barber DL, Wherry EJ, Ahmed R (2003) Cutting edge: rapid in vivo killing by memory CD8
T cells. J Immunol 171:27–31
7. Jia Y, Akache B, Deschatelets 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
8. Jungblut M, Oeltze K, Zehnter I et al (2008)
Preparation of single-cell suspensions from
mouse spleen with the gentleMACS Dissociator.
J Vis Exp (22):1029
558
Felicity C. Stark et al.
after injection into naı ¨ve animals. Variation in Target–Nontarget cell ratios such as 55:45 (or similar) are normal; it is not
necessary to discard “Target” cells to ensure that an exact equal
number of cells are injected. The most important criteria is to
maintain a consistency in the number of “Target” cells injected
within an experiment or preferably as a standard for all
experiments.
17. Cells will be injected at a concentration of 100 million cells/ml
(20 million cells/200 μl/mouse). “Target” cells and “Nontarget” cells are counted separately to take note of any extreme
differences in cell count. Any extreme loss of cells (>50%)
could be a result of CFSE staining toxicity and more care
would be taken during these steps.
18. Typically, mice are euthanized 24 h after cell injection; however, for logistical purposes, we euthanize at 20 h and still
observe the same extent of target cell killing.
19. If samples cannot be acquired immediately, cover the deep-well
plate at this step and refrigerate until ready (up to 4 h). Since
live splenocytes can readily reform clumps, it is best to filter the
cells immediately before acquisition by flow cytometry.
20. If a plate reader is not available or if there are only a few
samples, cells can be individually transferred to 5 ml roundbottom polystyrene tubes and acquired manually.
21. Flow cytometry data from our immunization studies are routinely analyzed using FlowJo™ 10 (BD Biosciences, Franklin
Lakes, NJ, USA). CFSE
+ cells are graphed and analyzed for
statistical significance using GraphPad
® Prism 8 (GraphPad, La
Jolla, CA, USA).
References
1. Hollingsworth RE, Jansen K (2019) Turning
the corner on therapeutic cancer vaccines. NPJ
Vaccines 4:7
2. Garc ¸on N, Di Pasquale A (2016) From discovery to licensure, the adjuvant system story. Hum
Vaccin Immunother 13:19–33
3. De Gregorio E, Caproni E, Ulmer JB (2013)
Vaccine adjuvants: mode of action. Front Immunol 4:214
4. Landi A, Law J, Hockman D et al (2017) Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP,
a STING activator or archaeosomes. Vaccine
35:6949–6956
5. Wooldridge L, Lissina A, Cole DK et al (2009)
Tricks with tetramers: how to get the most from
multimeric
peptide–MHC.
Immunology
126:147–164
6. Barber DL, Wherry EJ, Ahmed R (2003) Cutting edge: rapid in vivo killing by memory CD8
T cells. J Immunol 171:27–31
7. Jia Y, Akache B, Deschatelets 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
8. Jungblut M, Oeltze K, Zehnter I et al (2008)
Preparation of single-cell suspensions from
mouse spleen with the gentleMACS Dissociator.
J Vis Exp (22):1029
558
Felicity C. Stark et al.