B Proliferation assays for cell function

Summary

Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from "Compendium Immunology Laboratory Guide".

Operation method

B Cell Functional Reproduction Assay

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Basic program Anti-I g M and L P S-stimulated B cell proliferation Materials (see Appendix 1 for items with V)

Purified resting B cells (Unit 2.5) prepared by Percoll gradient centrifugation

V D M E M -10 Complete Medium

Goat anti-IgM (JacksonImmunoresearch) or bacterial LPS (E . Difco #011B 4)

0.lm C i/m l of [ ³ H ] thymidine dissolved in HBSS (Appendix 1) (20Ci/mmol/L ; Du PontN EN )

15 m l polypropylene tube (Falcon)

9 6-well flat-bottomed microtiter plate (Costar)

Collection system for direct collection of cells onto glass fiber slats (P H D Harvester, Cambridge Technologies)

Option 1 Polyclonal activation of B cells stimulated with 8-mercaptoquinoline (8-MG) Additional material (see basic program)

8- M G (Sigma)

0.Imol/L N a O H

2mol/L HCl

1. If 8-MG is used as the sole B cell activator, purified B cells between 50 % and 60 % Percoll are used instead of resting B cells in the 70 % Percoll zone (Unit 2.5).

2. Prepare 2 mmol/L 8-MG solution: dissolve 5 m g of 8-MG (Sigma) in 0.3 ml of 0-lmol/L NaOH, add 7.0 ml of DMEM-IO with additives, and adjust the p H to 7.0 with 2 mol/L HCl (15M1 ).

3. Add 0.05 ml of 2 mmol/L 8-M G alone or in combination with anti-Ig (0.5 mmol/L final concentration) to 96-well culture plates to a final volume of 0.2 ml per well as in step 3 of the basic protocol.

4 . Continue with steps 4 to 9 of the basic protocol.

Option 2 Stimulation of B-cell polyclonal activation with protein kinase C activator and calcium carrier Additional material (see basic program)

lmmol/L ethanol soluble PMA and PDBU (Sigma)

10 mmol/L ethanol-soluble indolactam (LC Services)

lmmol/L ethanol soluble indolactam (Calbiochem)

1 . D M E M -I O containing additives was used to prepare working solutions of P K C activator with calcium ion carriers (three dilution concentrations of each set):

0.04 to 4. Opmol/L P M A with P D B U

0.04~4. Ojumol/L indolactam

0.4~4. Ommol/L erythromycin

2. 50M1 P K C activator and 504 erythromycin were added to 96-well culture plates at each dilution (from Step 1) according to Step 3 of the basic protocol (final concentrations in culture wells were: P M A and P D B U, 0.01-1.0 Mmol/L; P M A and P D B U, 0.01-1.0 Mmol/L; P M A and P D B U, 0.01-1.0 Mmol/L).
indolactam, 0.01~1.0jtxmol/L; erythromycin, 0.1~I.Ommol/L).

3. Continue with steps 4 to 9 of the basic program.

Option 3 Polyclonal Activation of B Cells Stimulated with Glucose Sulfate and Poly (I : C) Additional material (see basic program)
Option 4 Quantification of cell count increase Additional material (see basic program for additional material, see Appendix 1 for items with n/)

V Phosphate Buffer Salt (P B S)

0.5mol/L E D T A prepared with P B S (ice cold)

Ice-cold 95 % ethanol

lmmol/L Hoechst 33342 (Sigma)

lmmol/L Pyronin Y (Sigma)

5 ml round-bottomed centrifuge tubes (Falcon)

Dual laser flow cytometer (e.g. FACScan, Becton Dickinson; Unit 4.2)

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