20161115 B cells: pre-array IVIg stimulation BIS

OBJECTIVES

To extract RNA from IVIg treated and non-treated stimulated and non-stimulated  B cells from healthy individuals.

INDIVIDUALS

4 CPT tubes from two healthy controls (Luis & Ana).

MATERIALS

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

– RNeasy Micro Kit (cat. no. 74004) Qiagen

PROTOCOLS

A) PBMCs isolation in CPT tubes

1) Centrifuge 20 min at 2790 rpm (1650g) at 18-25 ° C without brake. Samples must be processed within 24 hours after extraction. In the meantime, prepare:

A) 50 mL of B cell isolation recommended medium: PBS + 2% FBS + 1 mM

In a 50 mL Falcon, add: 1 mL of FBS* + 100 µL 0.5M EDTA** + 49 mL of PBS***

* previously tempered.

** at the culture room fridge, sterile.

*** at the culture room flux cabin.

B) 500 mL of culture medium: RPMI 1640 without FBS supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin.

In a stericup, previously tempered and cleaned with alcohol, add: 49 mL  RPMI, 0.5 mL  glutamine and 0.5 mL of the combo penicillin/streptomycin. Expiration date: 2-3 months after unless turbidity or any kind of growth is observed.

2) Invert the tube several times to resuspend the cells in the plasma.

3) Decant the supernatant into a Falcon tube.

4) Fill Falcon with SF to approx. 45 mL.

5) Centrifuge at 300g for 5 min with brake and acceleration at RT and remove supernatant without touching the pellet.

6) Resuspend the pellet by adding to 45 mL of SF.

7) Centrifuge at 300g for 5 min with brake and acceleration at RT and remove supernatant without touching the pellet.

8) Resuspend the pellet in 2 ml PBS +  2% FBS + 1 mM EDTA.

9) Perform cell count.

B) Bcell isolation using “EasySep negative selection Human B-cell enrichment kit” by Stemcell Technologies (#190549) (Manual protocol using the purple easysep magnet)

 1)Prepare cell suspension (500 μL – 2 mL) at a concentration of 5 x 10⁷ cells/mL  (up to 1 x 10⁸ cells) in the recommended solution (prepared in step 3A). Cells must be placed in a 5 mL (12 x 75 mm) polystyrene tube  to properly fit into the Purple EasySep Magnet.

2) Add the Human B Cell Enrichment Cocktail at 50 μL/mL of cells. Mix well and incubate at room temperature for 10 minutes.

3) Vortex the D Magnetic Particles for 30 seconds. Ensure that the particles are in a uniform suspension with no visible aggregates.

4) Add the D Magnetic Particles at 75 μL/mL of cells. Mix well  (Careful: place tap in order to vortex aoutside the flux cabin) and incubate at room temperature for 5 minutes.

5)Bring the cell suspension up to a total volume of 2,5 mL  by adding recommended solution. Mix the cells in the tube by gently pipetting up and down 2 – 3 times.

6) Place the tube (without cap) into the magnet. Set aside for 5 minutes.

7) Pick up the magnet, and in one continuous motion invert the magnet and tube, pouring off the desired fraction into a new 5 mL polystyrene tube. Leave the magnet and tube in inverted position for 2 – 3 seconds, then return to upright position.

Caution: do not shake or blot off any drops that may remain hanging from the mouth of the tube.

8) Perform cell count as described in previous steps.

C) Bcell culture and stimulation

1) Culture B cells at a density of 2,5·10⁶ cells/ml in complete medium: RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin.

Three conditions were assayed in the experiment (non-stimulated and non-IVIg-treated cells, stimulated and non-IVIg-treated cells and stimulated and IVIG-treated cells respectively), so the 2,5 mL suspension was divided in three tubes . To bring the concentration up to 2,5·10⁶ cells/ml, each eppendorf was centrifuged  at 300g 5 min and the pellet was resuspended in the appropriate volume of medium.

2) Stimulate due B cells  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 24 h at 37°C, 5% CO2.

* Bibliography (Lin et al. 2014) describes the use of 3 µg/ml CpG-B 2006 (Invivogen) solution, but due to pippeting difficulties, the use of a 6 µg/ml solution was preferred.

** Originally:

Class B (recommended by Lin et al./ B cell specific): 1 mg vial resuspended in 260 µL of water (conc: 500 µM). 5 µL aliquots are kept at -20ºC in  the culture room freezer. For the 500 µL assayed, 1µL of the 500 µM stock solution was added to the well.

*** Kept in the culture room fridge at a commercial concentration of 2,4 mg/mL. For the 500 µL assayed, 2µL of the 2,4 mg/mL stock solution was added to the well.

3) After 24 h, depending on the condition, add:

– non-stimulated and non-IVIg-treated cells: add 20 mg/ml  of a BSA solution (in complete medium RPMI 1640 (w/o FBS) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin)

– stimulated and non-IVIg-treated cells: add 20 mg/ml  of a BSA solution (in complete medium RPMI 1640 (w/o FBS) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin) + 6 µg/ml CpG-B 2006 (Invivogen) and 10 µg /ml Goat Anti-Human IgA + IgG + IgM

– stimulated and IVIG-treated cells: add 20 mg/ml  of a dialized (vs complete medium RPMI 1640 (w/o FBS) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin) IVIg preparation (Grifols)+ 6 µg/ml CpG-B 2006 (Invivogen) and 10 µg /ml Goat Anti-Human IgA + IgG + IgM

4) Culture for 24 h at 37°C, 5% CO2.

D) RNA isolation with the RNeasy Micro Kit

The RNeasy Micro Kit (cat. no. 74004) is shipped at ambient temperature. Store the RNeasy MinElute® spin columns and the RNase-Free DNase Set immediately at 2–8°C. Store the remaining components dry at room temperature (15–25°C). All kit components are stable for at least 9 months under these conditions.

Notes before starting

• If purifying RNA from cell lines rich in RNases or tissue, add either 10 μl β-mercaptoethanol (β-ME), or 20 μl 2 M dithiothreitol (DTT), to 1ml Buffer RLT before use. Buffer RLT containing DTT or β-ME can be stored at room temperature for up to 1 month.
• Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution.
• When processing <500 cells, carrier RNA may be added to the lysate before homogenization (see the RNeasy Micro Handbook for information).
• To prepare DNase I stock solution, dissolve the lyophilized DNase I in 550 μl RNase-free water. Mix gently by inverting the vial. Do not vortex. Store DNase I as single-use aliquots at –20°C for up to 9 months, or Store at 2–8°C for up to 6 weeks. Do not refreeze after thawing.

Protocol

1. Cells: Harvest a maximum of 5 x 105 cells, as a cell pellet, or by direct lysis in the vessel. Add 350 μl Buffer RLT and homogenize. Homogenize the lysate by vortexing 1 min.
2. Add 1 volume of 70% ethanol to the lysate, and mix well by pipetting. Do not centrifuge. Proceed immediately to step 3.
3. Transfer the sample, with any precipitate, to an RNeasy MinElute spin column in a 2 ml collection tube (supplied). Close the lid and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
4. Add 350 μl Buffer RW1 to the RNeasy MinElute spin column. Close the lid.Centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
5. Add 10 μl DNase I stock solution to 70 μl Buffer RDD. Mix by inverting the tube. Add the DNase I incubation mix (80 μl) directly to the RNeasy MinElute spin column membrane. Place on the benchtop (20–30°C) for 15 min. Add 350 μl Buffer RW1 to the RNeasy MinElute spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the collection tube.
6. Place the RNeasy MinElute spin column in a new 2 ml collection tube (supplied). Add 500 μl Buffer RPE to the spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
7. Add 500 μl of 80% ethanol to the RNeasy MinElute spin column. Close the lid, and centrifuge for 2 min at ≥8000 x g. Discard the collection tube.
8. Place the RNeasy MinElute spin column in a new 2 ml collection tube (supplied). Open the lid of the spin column, and centrifuge at full speed for 5 min to dry the membrane. Discard the flow-through and collection tube.
9. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube (supplied). Add 14 μl RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at full speed to elute the RNA.
10. Measure RNA content by Nanodrop quantification and freeze sample at -80ºC.

RESULTS