20180320:ODN & anti-IgG+M+A functionality test

OBJECTIVES

– To test whether the  available aliquotes of  ODN + anti G/M/A Ab  properly stimulate B cells. In comparison to the  20180316 experiment, we will test both stimuli individually and together.

MATERIALS

–  isolated B cells from one individual: individual ct Luis

–  “EasySep negative selection Human B-cell enrichment kit” (Ref.190549 Stemcell Technologies)

– CpG-B 2006 (Invivogen)

-Goat Anti-Human IgA + IgG + IgM (H+L; anti-Ig; Jackson Immunoresearch Laboratories)

-“IL-10 Secretion Assay – Detection Kit (PE), human ” kit Ref: 130-090-434, Miltenyi.

– anti-CD19-AF700 antibody (Ref.302226 BioLegend)

– LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Ref.L34957 Invitrogen)

PROTOCOLS

Note:  B cells were analized using fmo. No beads or IC’s were assayed in this experiment. Only the  Aqua+AF700+PE condition was assayed. This assay was conducted using the same settings as in previous experiments.

A) PBMCs isolation

PBMCs were isolated using CPT tubes as explained in previous experiments. 85.75 million cells were isolated. All of themwere used for B cell isolation and stimulation previous flow cytometry.

B) B cell isolation

Our starting material was a suspension  with 85.75 million cells. After the purification step, 2.96 million cells in the 2,5 mL suspension were found.

B) Bcell culture and stimulation

Four conditions were assayed in the experiment (ODN stimulated B cells; anti G/M/A Ab stimulated B cells;  ODN +anti G/M/A Ab stimulated B cells and non-stimulated cells ), so, for all individuals, the 2,5 mL suspension was divided in 4 tubes containing  0.74 millions each.

To bring the concentration  to 2.5·10⁶ cells/ml, each eppendorf was centrifuged  at 300g 5 min and the pellet was resuspended in 296 mcL medium. Due tubes were stimulated as previously described  (with 6 µg/ml CpG-B 2006 (Invivogen) and 10 µg /ml Goat Anti-Human IgA + IgG + IgM*** (H+L; anti-Ig; Jackson Immunoresearch Laboratories) for 48 h at 37°C, 5% CO2 )

In all cases, cells were cultured in 5 mL (12 x 75 mm) polystyrene tubes w/o adding Monensin. Since those were same tubes in which the following staining and flow cytometry analysis were performed, and thus no cell loss was expected, after 48h no cell counting was conducted.

C) ANÁLISIS DE LA MORFOLOGÍA CELULAR

De cada condición, reservamos 75 mcL de los 296 mcL totales para analizar la morfología de las células mediante tinción de Wright-Giemsa. El resto de muestra se destina a citometría como en ensayos anteriores.

1. Separar 75 mcL de muestra. Lavar con PBS dos veces (400 g 5 min) para eliminar el FBS de la preparación (éste forma cristales con la tinción de Giemsa)
2. Añadir volumen correspondiente al porta (100 mcL)
3. Cytospin 5 min 400 rpm (aceleración y freno medio)
4. Dejar secar al aire mínimo 20-30 minutos (recomendable 1 h)
5. Fijar las células con metanol 5 minutos a TA
6. Dejar secar al aire
7. Tinción con Wright’s eosin methylene blue solution (Sigma 1.013830500) diluído ½ con tampón fosfato 15M (Eder): 4 minutos a TA
8. Lavar por inmersión en H2Od dos veces
9. Tinción con Giemsa’s azur eosin methylene blue solution (Sigma 1.092040500) al 15% en tampón fosfato 15M (Eder): 4 minutos a TA
10. Lavar por inmersión en H2Od dos veces
11. Dejar secar al aire
12. Añadir una gota de medio de montaje (Surgipath Micromount Mounting Medium) y poner cubre.

D) IL-10 secretion analysis by FC

Once cells were harvested from culture:

1. Use one million total cells in a 2 ml closable tube per sample.  Note: For larger cell numbers, scale up all volumes accordingly. For less than one million cells, use same volumes.

2. Wash cells by adding 1-2 ml of cold buffer, centrifuge at 300xg for 10 minutes at 4-8°C, pipette off supernatant completely.  Note: Do not remove supernatant by decanting. This will lead to cell loss and incorrect incubation volumes.

3. Resuspend cell pellet in 90 μl of cold medium per million total cells.

4. Add 10 μl of IL-10 Catch Reagent per million total cells, mix well and incubate for 5 minutes on ice.

5. Add warm (37°C) FBS-free medium  w/o stimuli to dilute the cells accoding to the following proportions.

– If less than 5% IL-10 secreting cells are expected: add 1 mL of warm medium per million cells. (our case)

– If more than 5% IL-10 secreting cells are expected: add 10 mL of warm medium per million cells.

6. Incubate cells in closed tube for 45 minutes at 37°C under slow continuous rotation or turn tube every 5 minutes to resuspend settled cells.

7. Put the tube on ice.

8. Wash the cells by filling up the tube with cold buffer, centrifuge at 300xg for 10 minutes at 4-8°C. Pipette off supernatant completely. Note: If the volume of the cell suspension was higher than the volume of added buffer, repeat wash step.

9. Resuspend cell pellet in 90 μl of cold buffer per million  cells.

10. Add 10 μl of IL-10 Detection Antibody (PE) per million cells.

10. Add additional staining reagents: 2 µL of the CD19-AF700 antibody per million cells.

11. Mix well and incubate for 10 minutes on ice.

12. Wash cells by adding 2 ml of cold buffer, centrifuge at 300xg for 10 minutes at 4-8°C, pipette off supernatant.

13. Add 1 mcL of the reconstituted fluorescent reactive dye (Aqua Live/Dead Stain) per million cells to 1 mL of the cell suspension and mix well. Incubate at room temperature or on ice for 10 minutes, protected from light.

14. Proceed to analysis. Acquire 200.000 viable cells from each sample.

RESULTS

CONCLUSIONES

Tanto el análisis morfológico de las células al microscopio como la citometría evidencian que el estímulo aplicado a las células B no funciona: se aprecia una gran mortalidad en ambos experimentos así como una falta de diferenciación celular (comparar con resultados del experimento 20170124).