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In vitro B cell stimulation
Last updated date: Jul 2, 2020 Views: 1876 Forks: 0
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Materials:
- Mouse splenocytes or lymphocytes
- C10 media:
- 500ml RPMI
- 50ml heat inactivated FCS
- 5ml Penicillin, Streptomycin, L-glutamine
- 5ml HEPES
- 5ml NEAA
- 5ml Na-pyruvate
- 500ul BME.
- Anti-IgM: AffiniPure F(ab')₂ Fragment Goat Anti-Mouse IgM, µ Chain Specific, JacksonImmunoResearch 115-006-020
- 96-well round bottom plate, Corning 3799
- 40uM filter, Corning 431750
- FACS buffer
- 500ml PBS w/o Ca+, Mg+
- 5ml Penicillin, Streptomycin, L-glutamine
- 2ml 0.5m EDTA
- 10ml heat inactivated FCS
- PBS
- 4% PFA
- Live/dead APCe780, Invitrogen L10119
2X anti-IgM titration:
- Prepare a two-fold serial dilution of anti-IgM in C10 media starting with a concentration of [12.8ug/ml] down to [1.6ug/ml], for final concentrations of 6.4, 3.2, 1.6, and 0.8 [ug/ml].
Methods:
- Harvest LN (or spleen) into C10 media
- Single cell suspension through 40uM filter in 10ml C10
- Spin 1500rpm/5min/4˚C, dump supernatant
- Resuspend LN in 2ml C10; If splenocytes, need to use ACK for red cell lysis (RT 5 min, wash, spin as above and then resuspend in 2ml C10)
- Dilute (1:50) in C10 (10ul + 490ul C10), count cells
- Adjust total viable cell concentration to 5x106cells/ml in C10
- Plate 100ul cells/well (5x105cells/well), save remainder on ice for pre-stain
- Add 100ul C10 +/- anti-IgM according to assay design (or other stimuli if appropriate) soluble
- Incubate in a 37˚C humidified chamber with CO2 overnight (time point can vary depending on the readout 6-18h)
Stains to detect surface activation markers:
- Transfer cells to 96-well v-bottom plate,
- Spin 2000rpm/2min/4˚C, dump sup
- Wash w/ 200ul cold PBS, spin 2000rpm/2min/4˚C, dump sup
- Resuspend with 100ul live/dead APC-e780 (1:1000) in PBS
- Incubate on ice/20min/dark
- Spin 2000rpm/2min/4˚C, dump sup
- Wash w/ 200ul FACS, spin 2000rpm/2min/4˚C, dump sup
- Resuspend w/ 50ul stain, sit on ice/15min/dark
- Spin 2000rpm/2min/4˚C, dump sup
- Wash w/ 200ul FACS buffer, x2
- Add 100µl 4% PFA to FACS tubes
- Resuspend cell pellets in 100µl FACS buffer, transfer to tubes containing PFA
| Sample stain (may include CD23 if staining splenocytes) | ||
Stain | Dil. | |
| APC | - | - |
| PE-Cy7 | CD69 | 1:200 |
| PB | B220 | 1:200 |
| APCe780 | Live/dead | - |
| fcblock | fcblock | 1:250 |
| FACS | - | - |
Copyright: Content may be subjected to copyright.
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Zikherman, J and Mueller, J L(2020). In vitro B cell stimulation. Bio-protocol Preprint. bio-protocol.org/prep363.
- Noviski, M., Mueller, J. L., Satterthwaite, A., Garrett-Sinha, L. A., Brombacher, F. and Zikherman, J.(2018). IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate. eLife. DOI: 10.7554/eLife.35074
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