In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.
The protocol for the generation of murine CD40-activated B cells (mCD40B) from splenocytes is divided into two parts: Part A demonstrates the preparation of murine CD40 ligand (CD40L) expressing HeLa cells (tmuCD40L HeLa), which will be used as plate-bound feeder cells. Part B describes the actual murine CD40-B culture.
A. Preparation of feeder cells (tmuCD40L HeLa)
The tmuCD40L HeLa is an adherent human epithelial cell line, which should never become completely confluent. The cells are therefore splitted twice per week. Culturing over more than 6 weeks is not recommended.
B. Murine CD40-B cell culture
I. Preparation of splenocytes for CD40-stimulation (day 0):
Please note: before you continue ascertain that feeder cells are adherent. Always add fresh solutions of murine interleukin-4, β-Mercaptoethanol and cyclosporin A to the growth medium immediately before use.
II. Subculture of mCD40B cells (day 3 and then twice a week):
C. Trouble-shooting: What if CD40-B cells do not grow?
Figure 1a.
Figure 1b.
Figure 1d.
Figure 2.
Modifications of this protocol are possible, especially regarding the origin of CD40-stimulus and the type of mouse strain used, also yielding in efficient generation of murine CD40-activated B cells. Ahmadi et al. have shown similar results for mouse fibroblasts transfected with murine CD40-Ligand. In this context presentation of xeno-antigen can be excluded with certainty, but intensive studies regarding security and toxicity in vivo gave no signs for inflammation or auto-immunity reaction in this setting using human transfected HeLa cells. However, the best alternative represents a soluble murine CD40-Ligand with activating and proliferating activity at the same time, which is to our knowledge not commercially available.
This protocol also works for lymphocytes from 129S2/SvPas mice (haplotype H-2b), but has not been evaluated for further mouse strains so far. Furthermore differences in adjustment of cell density, cytokine concentration, duration of cyclosporine A treatment, cell culture media and dishes may also lead to efficient CD40-activation of murine B cells with similar proliferation rates. However, intensive work has been spent on optimizing this system maximizing activation and proliferation rates resulting in the present system.
The authors have nothing to disclose.
We kindly thank Dr. Clemens Wendtner for providing tmuCD40-Ligand-HeLa cell line.
A. Preparation of Media:
Feeder cell wild type medium | Feeder cell selection medium | ||
Reagent | Concentration | Reagent | Concentration |
RPMI 1640 | RPMI 1640 | ||
L-Glutamine | 300 μg/mL | L-Glutamine | 300 μg/mL |
FBS | 10 % | FBS | 10 % |
HEPES | 10 mM | HEPES | 10 mM |
Gentamycin | 15 μg/mL | Gentamycin | 15 μg/mL |
Hygromycin B | 0.2 mg/mL |
mCD40-B washing medium | mCD40-B culture medium | ||
Reagent | Concentration | Reagent | Concentration |
D-MEM | |
D-MEM | |
L-Glutamine | 580 μg/mL | L-Glutamine | 580 μg/mL |
Glucose | 4.5 mg/mL | Glucose | 4.5 mg/mL |
HEPES | 10 mM | HEPES | 10 mM |
Gentamycin | 15 μg/mL | Gentamycin | 15 μg/mL |
MEM | 0.1mM (1x) | ||
FBS | 10% |
B. Miscellaneous Reagents:
Reagent | Company | Order No |
RPMI 1640 | Invitrogen Gibco® | REF 21875 |
D-MEM | Invitrogen Gibco® | REF 41965 |
Dulbecco´s PBS (10x) | PAA | Cat No H15-011 |
Gencin® | Delta Select | Art No 7395800 |
FBS | LONZA | Cat No DE14-802C |
HEPES Buffer | PAA | Cat No S11-001 |
MEM NEAA | Invitrogen Gibco® | REF 11140 |
Hygromycin B | PAA | Cat No P02-015 |
Recombinant Murine Interleukin-4 | Immunotools | Cat No 12340045 |
Cyclosporin A | Novartis | Cat No NDC 0078-0109-01 |
Trypsin/EDTA (10x) | Invitrogen Gibco® | REF 15400-054 |
C. Miscellaneous Supplies:
Reagent | Company | Order No |
Sterile pipette tips | Sarstedt | |
6-well plate | NUNC | Cat No 140675 |
50mL conical tube | BD Falcon™ | Cat No 352070 |
Tissue culture flask | Sarstedt | Cat No 83.1813.002 |