Human Correlation Protocols

Ficoll Paque Preparation of Human Blood Mononuclear Cells

Human Peripheral Blood Mononuclear Cell Preparation

 

Typical yield is 1-2E6 PBMC per ml blood. For example if WBC is 8000 per µl, and 40% of the cells are PBMC, ~3E6 PBMC might be recovered per ml blood.

ACD is prepared by bringing 22 g trisodium citrate (dihydrate, f.w. 294) 8 g citric acid (monohydrate, f.w. 210) and 24.6 g dextrose (monohydrate, f.w. 198) up to 1 liter with pyrogen-free distilled water (Sterile water for irrigation, B. Braun). Use fresh, newly opened reagents to avoid endotoxin contamination. Final pH should be ~5.  Filter using 0.2 µm filter stand, Corning 431098 and aliquot into sterile 15 ml tubes, Corning 430055.

Isolation and Culture of Mononuclear Cell Subsets

T cells (CD4 and CD8), NK cells and B lymphocytes are prepared using positive or negative selection with magnetic beads. Cells that are not required for immediate use are cryopreserved in 10% DMSO (ATCC 4-X-5) in heated fetal bovine serum (Hyclone Defined FBS). Generally CD4, CD8 and NK cells are isolated using Miltenyi Microbeads as described in the product literature. B cells are prepared using negative selection.

T lymphocytes are cultured in DMEM +10 heated FBS + 50 U IL-2/ml and Dynabeads (Invitrogen, CD3/CD28 T cell expander) at 1:1, bead:cell. When cells are cultured immediately after isolation from PBMC, or when cells are thawed from cryopreserved stocks, 4 hours culture with IL-2 and Dynabeads is sufficient to activate cells for efficient Lentiviral transduction.

B lymphocytes, either freshly prepared or thawed from cryopreserved stocks, are cultured in DMEM + 10% heated FBS + CpG (e.g. ODN 2006, 2.5 µg/ml), IL-1 (100 U/ml), anti-human Ig (IgG, IgM, IgA, H+L, 2 µg/ml), anti-CD40 (e.g. clone HB14, 0.1 - 1 µg/ml), and with or without IL4 (20 ng/ml) and IL-10 (10 ng/ml). Soluble anti-CD40 antibody appears to work as well or better than anti-CD40 immobilized on beads (Dynal M450 Epoxy). The B cells are maintained in culture for 4 days prior to transduction with packaged Lentivirus.

Lentiviral shRNAmir Procedures

Amplification and storage-


Plasmid Prep-

(A 5 ml culture of shRNAmir construct is sufficient to produce 5-10 µg of plasmid DNA.)


Restriction Digest of GIPZ-


Transfection- (to evaluate the quality of the clones)

Protocol assumes transfection of shRNAmir plasmid DNA into HEK293T cells in 24-well clusters using medium that does not contain serum or antibiotics.



Separate reporter transfection is not required when construct already contains a reporter (e.g. GFP, luciferase and/or β-gal.) Use carrier-DNA such as pUC19 or pBluescript plasmid as needed to maintain optimal DNA/Arrest-In ratios.

Packaging Lentivirus-

Use the Trans-Lentiviral shRNA packaging System (TLP4614 or-15) to generate a replication-incompetent HIV-1-based lentivirus which may be used to deliver and express the gene of interest or shRNAmir in either dividing or non-deviding mammalian cells.

The kit provides sufficient packaging mix and Arrest-In for 10 packaging events in 100 mm plates.

Kit contents:


Transfection and Virus production-

(optimized for transfection of shRNA plasmid DNA into TLA-HEK293T cells in 100 mm tissue culture dishes using medium without serum or antibiotics.)


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Caveats:



Evaluation of Knockdown






Cell Lines, Media and Reagents:

DMEM (high glucose)

10% FBS (heated)

1% L-Glutamine/Pen/Strep 100x stock

Starting cells from frozen stock-




Microbiological Reagents

Note: Open Biosystems recommends low-salt/rich medium for production of plasmid, while Qiagen recommends standard LB (not enriched) broth with high salt (10 g NaCl/liter) for optimum plasmid production. However, because Zeocin is salt-sensitive, Invitrogen recommends selective growth in low salt medium (5 g NaCl/liter). Generally we use the Luri Bertini Lennox, unsupplemented as shown below (also checking pH to ensure Zeocin stability).

2X-LB Broth (high nutrient, low salt, from Open Biosystems handbook)-

Antibiotic Stocks-

Zeocin (Invitrogen R250-01) sterile 100 mg/ml stock is stored at -20°C. Protect from light, and thaw on ice. Freshly autoclaved media should be cooled to 55°C or lower, and Zeocin added to final concentration of 25 µg/ml for selection in bacterial cultures (e.g. 12.5 µl per 50 ml medium). Because Zeocin is salt and pH sensitive, the recommended growth medium is low-salt LB (e.g. Invitrogen LB broth base, Lennox, 12780-052, which contains 10g Tryptone, 5g Yeast extract and 5 g NaCl per liter). Weigh the powder (20g/liter) dissolve, and check the pH (should be ~pH 7.5). When plates are needed, add 15g/liter of agar (Bacto 214010) microwave until agar is melted and dissolved (just beginning to boil). Autoclave 20 minutes at 15 psi, 121°C.

Carbenicillin (e.g. Sigma C3416): Prepare a 10 mg/ml stock (100x) in sterile water and pass through a 0.2 µm filter. Aliquot and store at -20°C. Dilute into broth or agar-medium (cooled to 55°C) to a final concentration of 100 µg/ml.

Luria Bertani medium (Lennox, unsupplemented) per Invitrogen Recommendation for Selective Growth-

The standard LB medium contains 10 g peptone, 5 g yeast extract and 5 g NaCl per liter.



Qiagen- Culture and Plasmid Purification

Though Qiagen recommends Luria Bertani medium (e.g. LB Miller, with 10g peptone, 5g yeast extract and 10g NaCl per liter) we use the Lennox low salt (5g/liter) broth base to improve the stability of the Zeocin selective antibiotic. Single colonies should be grown under selection followed by production of inoculum culture and finally a production culture grown to full-log 3-4E9 cells per ml or about 3 g wet weight per liter).

Grow bacteria-


Prepare reagents-


Extract and purify DNA (Qiagen Plasmid Midikit, Cat. No. 12143)-


DNA Quantification-

Note, A260 for purified DNA should be 1.0 for a 50 µg/ml solution at neutral pH.


Agarose Gel Electrophoresis-

Gel preparation-