Ficoll Paque Preparation of Human Blood Mononuclear Cells

Human Peripheral Blood Mononuclear Cell Preparation

• Collect blood using ACD anticoagulant (~140 ml, using the ratio 15ml ACD:100 ml blood)
• Transfer to 225 ml bottles (Falcon 352075) and dilute with equal volume of DPBS (Gibco 14190)
• Mix and transfer 20 ml aliquots to 50 ml tubes (Corning 430290, 16 tubes total)
• Transfer the amount of Ficoll Paque (GE Healthcare 17-5442-03) needed for the preparations to 50 ml tubes to avoid contaminating stock (e.g. three tubes of ~41 ml ea).
• Using a spinal needle (18G, 3.5 in.) and 10 ml syringe underlay the blood in each tube with 7.5 ml ficoll.
• Spin 400 x g (RT6000 1600 rpm) for 35 minutes at 20°C).
• Draw off upper plasma/platelet layer using a sterile transfer pipet (discard plasma)
• Use a fresh sterile pipet to transfer the mononuclear cell layer to a sterile 225 ml conical tube. (Try to minimize platelet/upper layer and ficoll/lower layer contamination of the mononuclear cells at the boundary.)
• Limit the pooled material to 40 ml or less per 225 ml tube, using additional tubes as needed (to allow dilution of the carry-over ficoll).
• Add ~185 ml DPBS + 5% heated FBS to each of the 225 ml tubes (filling the tube to the neck to obtain maximum dilution of the cell suspension and any carry-over ficoll).
• Cap and spin 1600 rpm at 20°C, 20 minutes. Take a few drops of the supernatant to examine on a slide or counting chamber for residual supernatant cells. Discard the supernatant (unless large numbers of cells warrant additional centrifugation).
• Shake the tubes to break up pellets, resuspend and pool the cells in 100 ml DPBS + FCS. Use a sterile pipet tip to transfer 20 µl to a chamber, and count cells. Aliquot cells as need for CD4 / CD8 / NK cell / B lymphocyte / monocyte preps. See separate protocol for lymphocyte subset preparation. Note: Cells yield = # cells per large square x 1E4 x ____ml.
• Spin the excess cells 1600 rpm at room temperature for 20 minutes. Resuspend the cell pellet in sufficient 10% DMSO/heated FCS to have 1E7 cells per cryovial.

 

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-

• Remove GIPz clones from -80°C storage and inoculate 2 µl primary stock culture per 2 ml LB containing 25 µg zeocin, 100 µg/ml carbenicillin and 8% glycerol
• Incubate with shaking at 30°C for 16-20 hours.
• Freeze at -80°C for long-term storage.

Plasmid Prep-

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

• Thaw a GIPz glycerol stock and pulse vortex to suspend the E. coli.
• Transfer a 10 µl inoculum of the glycerol stock to 5 ml of low-salt LB containing 100 µg/ml carbenicillin. (Return glycerol stock to -80°C.)
• Incubate at 37°C for 16 hours with vigorous shaking. (If larger cultures are needed, use this 16-hour culture as inoculum for a larger culture.)
• Centrifuge the culture, and recover the bacterial pellet for plasmid DNA preparation.
• Run 200 ng (generally 3-5 µl) of the plasmid DNA on a 1% agarose gel. (The pGIPz + shRNAmir is 11,744 bp.)

Notes:

• To avoid formation of recombinants, keep all incubation times as short as possible, and avoid subculturing.
• The bacteria can be grown at 37°C for template prep or sequencing. However, for archival replication grow all pGIPZ clones at 30°C in low-salt LB with zeocin and carbenicillin.

Restriction Digest of GIPZ-

• Using filter pipette tips and sterile conditions make the following additions to a sterile PCR thin-wall tube:
• Sterile nuclease-free water x µl
• Restriction enzyme buffer, 10-x 1 µl
• BSA (10 mg/ml stock) 1 µl
• DNA sample (80-240 ng in d.w. or TE) x µl
• Restriction enzyme, 20 U 0.25 µl
• Final volume 10 µl
• Mix gently by pipetting.
• Incubate in thermal cycler at 37°C for 2 hours.
• Load 10 µl of the digested sample on a 1% agarose gel.

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.

• Warm the Arrest-In transfection reagent to ambient temperature (~20 minutes on the bench) and mix by vortexing or inversion prior to use.
• Maintain sterile conditions working with the DNA and Arrest-In as the mixtures will be added to cells.
• One day before transfection plate the HEK293 cells at 5E4/well in a 24-well cluster using complete medium with serum and antibiotics.
• On the day of transfection prepare the DNA/Arrest-In complexes.
• Set up two microfuge tubes for each well to be transfected:

1. Dilute 500 ng shRNAmir into a final volume of 50 µl serum-free medium.
2. Add 2.5 µg (2.5 µl) of Arrest-In to 47.5 µl serum-free medium.

• Transfer the diluted DNA from tube 1 to the diluted Arrest-In (tube 2); mix rapidly, and incubate for 20 minutes at room temperature. (Note: 1:5 DNA:Arrest-In ratio.)
  

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.

• Add an additional 150 µl serum-free medium to each of the tubes containing the DNA/Arrest-In complexes, and mix gently. Aspirate the growth medium from the HEK293 cells, and add the 250 µl DNA/Arrest-In complexes. Incubate for 3-6 hours in a CO₂ incubator at 37°C.
• After the initial incubation add 250 µl additional growth medium containing 2X serum (e.g. 20 % serum) and return cells to the incubator. (If toxicity of the transfection complexes is a problem, remove the medium from the cells after the initial incubation, and replenish with 500 µl medium with 10% FBS.)
• Observe cells 48-96 h after transfection for evidence of reporter expression. Then evaluate cells for reduction in gene/reporter by qRT-PCR, or western blot.

(Optimal duration of transfection-to-analysis will depend on cell type, gene of interest, stability of the mRNA and/or protein.)

• When selection of stably transfected cells is desired transfer the cells to medium containing puromycin, but this selection process should be delayed at least 48 hours. The puromycin working concentration varies from 1-10 µg/ml depending on the cell line. The lowest concentration should be used that can be shown to kill 100% un-transfected cells within 1-4 days.

(Optimum conditions for transfection can be determined by varying DNA:Arrest-In ratio, DNA concentration and cell confluency.)

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:

• Trans-Lentiviral packaging mix containing optimized mixture of five packaging plasmids (pTLA1-Pak, pTLA1-Enz, pTLA1-Env, pTLA1-Rev and pTLA1-TOFF).
• TLA-HEK293T cells for production of lentivirus following co-transfection of the transfer plasmid and the packaging plasmids. The TLA cell line stably expresses SV40 large T antigen and facilitates production of high viral titers.
• Arrest-In proprietary lipo-polymeric transfection reagent which optimizes uptake and transfection of shRNA plasmid DNA into the nucleus of eukaryotic cells
• pGIPZ non-silencing control vector for use as expression control to generate non-silencing lentiviral stock to optimize expression conditions in the mammalian cell line of interest.
• pGIPZ cloning vector containing Turbo GFP reporter and also elements required to allow packaging of the expression construct into virions (i.e. 5' and 3' LTRs and Ψ packaging signal).

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.)

• One day before transfection plate the TLA-HEK293T cells at 5.5E6/100mm dish in 10 ml complete medium with serum and antibiotics. This provides an initial cell density of 1E5/cm².
• On the day of transfection prepare DNA and Arrest-In as described above except that for each plate, dilute 37.5 µg DNA (26 µl packaging mix containing 28.5 µg DNA and 9 µg pGIPZ transfer vector) into 1 ml final volume serum free medium (in a microfuge tube), and dilute 187.5 µl Arrest-In (1 mg/ml stock) up to 1 ml in serum-free medium (in a 14 ml polypropylene tube, Falcon 352059).
• Transfer the diluted DNA to the tube containing the diluted Arrest-In, mix rapidly, and then incubate for 20 minutes at room temperature.
• Add an additional 3 ml of serum-free medium to each of the tubes containing the DNA/Arrest-In complexes, mix gently, remove the growth medium from the cells, and add the complexes to the cells. Return the cells to the CO₂ incubator at 37°C. The recommended incubation time is 3-6 hours, but we find that 3.5 hours is optimal for exposure of HEK293 cells to the transfection mixture.
• After the 3.5 hour exposure of the HEK cells to complexes gently aspirate the transfection mixture and replace with 12 ml standard culture medium.
• After 48 hours examine the cells microscopically for TurboGFP expression.

Harvest the virus-containing supernatants 48 post-transfection by removing the medium for storage in a 15 ml, sterile conical tube. (Fresh medium could be added to the plates for an additional 24 hour of virus production, but we find that titers are substantially reduced in the 48-72 hour supernatant and not worth collecting.)

Caution: The viral particles in the supernatant are infectious. Use recommended procedures for BL-2 organisms.

Virus collected 48 hours after transfection may have sufficient titer to use directly on target cells. However we find that results are substantially improved when the virus is purified and concentrated by ultracentrifugation. When HEK293 cells are exposed to transfection mixture only 3.5 hours, the cells remain relatively healthy and adherent so that when the supernatant is collected at 48 hours, low speed centrifugation is not necessary to remove cellular debris, and the supernatant can be directly filtered (0.45 µm, Millex-HV Durapore PVDF membrane, SLHV033RS). It is preferable to proceed with ultracentrifugation as soon as possible after collection of the supernatant, though at 4°C the packaged virus is reasonably stable for 24 hours. Avoid a freeze/thaw cycle at this point if possible.

• Optimal materials for ultracentrifugation include: SW32Ti rotor (accommodating 6 x 38.5 ml), adapter for conical tubes (Beckman 358156), polyallomer thinwall konical^TM 25 ml and DPBS (Gibco 14190). Autoclave centrifuge tubes (121°C, 15 psi, 15 minutes), and prepare 20% sucrose in DPBS (sterilized by 0.2 µm filtration).
• Working in the laminar flow hood, pipet 1.5 ml sterile 20% sucrose into the centrifuge tubes and overlay with 22-23.5 ml of packaged Lentivirus supernatant.
• Load the tubes into the SW32 rotor and centrifuge at 25,000 rpm, 4°C for 1.5 hours.
• Pour the supernatant into a waste beaker, and keeping the tube inverted, use a P200 with sterile tip to remove the last drops of liquid from the tube. Add DPBS (1/100 of the original volume), typically 220 µl per 25 ml conical tube. Resuspend the virus by thumping the bottom of the tube for ~0.5 minutes followed by pipetting up and down 6-12 times. Pool virus recovered from duplicate tubes, prepare 50 µl aliquots in 0.5 ml Sarstedt tubes, and store at -80°C.

Viral stocks should be stable for ~ 1 year at -80°C. Store as multiple aliquots to avoid freeze/thaw cycles which result in loss of viral titer.

Transduction and Titering-

Determine the titer of the silencing lentiviral stock as well as non-silencing controls. Primary titrations are done in HT-1080 cells or TLR-HEK293T cells and subsequently should be also performed using the cells that will be used in experiments (e.g. primary cultures of lymphocytes or monocytes).

• One day before transduction seed a 24 or 48-well cluster with target cells (e.g. TLA-HEK293T cells) at 5E4 cells/well in DMEM containing 10% FBS and 1% Pen/Strep. On the following day the cells should not be more than 40-50% confluent.
• Prepare dilutions of the viral stock using a round bottom 96-well plate using dilution medium (DMEM containing 0.5% FBS and 8 mg/ml polybrene. (The polybrene is a cation which coats the viral particles, conferring a net positive charge to counteract the negatively-charged cell surface.)

• Add 80µl of the dilution medium to columns 1-8 of as many rows as needed (using one row to titrate each viral stock).
• Add 20 µl of each thawed virus stock to the designated well in column 1 (1/5 dilution). Pipet up and down 10-15 times, and discard tip.
• Using a fresh tip(s), transfer 20 µl from the well(s) in column 1 to the corresponding well in column 2. Pipet 10-15 times and discard tip(s).
• Continue the serial dilution(s) from the well(s) in column two through column 8, yielding a final dilution of ~1.390K.
• Allow the freshly prepared virus dilutions to stand at room temperature for 5 minutes.
• Label the 24-well cluster containing the mammalian target cells, marking one row for each viral preparation to be tested.
• Remove the culture medium from the 24-well plate, and add back 150 µl of transduction medium (DMDM + 0.5% FCS but without polybrene).
• Transduce the cells by adding 25 µl of diluted virus from the 96-well plate, e.g. using dilutions from columns 2-7, 1-6 or 3-8 depending on the estimated viral titer.
• Incubate the transduced cultures overnight at 37°C in a CO₂ incubator.
• After the overnight incubation add 0.8 ml DMEM containing 10% FBS and 1% pen/strep, and return to incubator for 48 hours.
• Using the fluorescence microscope, count the numbers of TurboGFP expressing cells (or colonies of cells, as proliferation should have occurred).
• The calculated viral titer, TU/ml = Number of GFP-positive cell/colonies x dilution x 40.

When the titer of the viral preparation is determined, the optimal MOI will need to be established for the mammalian cell line of choice. The optimal MOI will depend on several variables, including the nature of the mammalian cell line (e.g. dividing or non-dividing) its transduction efficiency, the gene of interest, etc. Initial experiments should be carried out using a range of MOIs (e.g. 0, 0.5, 1, 2, 5, 10, 20 etc). To achieve single copy knockdown an MOI of 0.3 should result in fewer than 4% of the cells receiving more than one insert.

Puromycin Kill Curve-

• Plate 5-8E4 cells per well in 24-well plates(or proportionally fewer cells in 48-well clusters), and incubate overnight at 37°C in a CO₂ incubator.
• Prepare medium containing various concentrations of puromycin over the range 0-15 µg/ml, and replace the growth medium in the 24-well cluster with the medium containing various concentrations of puromycin.
• Return the clusters to the incubator, and every 2-3 days replace the culture medium with freshly-prepared selective medium.

• Monitor the cells daily, and count the numbers of surviving cells.

• The minimum antibiotic concentration to use is the lowest concentration that kills 100% of the cells in 1-4 days from the start of antibiotic selection.
• Using a similar approach set up kill curves for the virus transduction targets (e.g. T or B lymphocytes) using standard cell densities in 48-well or 96-well clusters (e.g. 6E5 cells per well in 48-well clusters) in the presence of appropriate agonists.

Viral Transduction Followed by Puromycin Selection-

• Plate cells, and incubate overnight (typically 5-8E4 293 cells/well in 24-well cluster, adjusting culture area and volume as needed).

• After the overnight incubation remove the medium and add the virus to the optimum MOI in a sufficient volume of serum-free medium to cover the cells.
• This may be a small volume of concentrated virus + fresh serum-free DMEM or a larger volume of viral-containing unconcentrated supernatant from HEK293 cells.
• Typical final volume would be ~175-200 µl in a 24-well cluster. Use 1 ml medium per well in a 6-well cluster.
• Transduction efficiency may be improved substantially by addition of Polybrene. The Polybrene may be added directly to the virus supernatant before mixing with transduction reagent, or the Polybrene may be added to the cell culture immediately after addition of virus. Usual final concentrations are 4-10 µg/ml.
• When cells have been shown to resist viral transduction, centrifugation may improve efficiency. After addition of virus and polybrene to the cluster, place the cluster in a centrifuge carrier and spin in the RT600/H-1000B rotor. Cell cultures have tolerated 600-1000 x g for 30 minutes at 20°C.
• When virus-containing supernatant is yellow/acidic/metabolically depleted, addition of fresh medium may be helpful. For example, 6-8 hours after transduction add an additional 1 ml of complete medium (DMEM + 10% serum and pen/strep) and return the clusters to the incubator.
• At 48 hours post transduction, replace the growth medium with complete medium containing puromycin (as indicated in the above kill curve), and return the cluster to the incubator.
• Replace the medium every 2-3 days with freshly made selective medium.
• Monitor the cells daily to observe percentage of surviving cells as well as the level and percentage of GFP positive cells. Eventually all of the surviving cells should be GFP positive on or around day three of selection.

The puromycin concentration should be chosen relative to the MOI used for transduction. When higher MOIs are used more copies of the shRNAmir and puromycin resistance gene will be expressed per cell, and higher concentrations of puromycin will be tolerated by the cell culture. Accordingly, the puromycin concentration can be adjusted to select for the desired population, though the puromycin concentration should always exceed the minimum concentration indicated in the kill curve.

Caveats:

• Generic HEK293 cells can be used for packaging, but the TLA-HEK293T cell line from Open Biosystems (which stably expresses the SV40 large T antigen) should provide higher virus titers. The Invitrogen HEK293FT cells are comparable but will need to be grown in the presence of Geneticin (G418).
• Always use purified plasmid DNA, and determine the concentration by OD260.
• Always carefully mix the transfection reagent and also the DNA/Arrest-In complexes before addition to cells.
• Transfection cytotoxicity may be minimized by optimizing the DNA:Arrest-In ratio.
• Perform transfection in serum/antibiotic-free conditions unless cell viability requires serum.
• High protein expression levels can cause cytotoxicity.
• After transfection with the transfer plasmid and packaging mix the HEK293 cells will began to express VSV-G glycoprotein which causes the cells to fuse, forming multinucleated syncitia.
• Use cells that have a known/documented history regarding passage number and growth density. In general better results will be obtained with low-passage-number cells. Cells should be freshly plated at appropriate density on the day before transfection.

Evaluation of Knockdown

• Extract cellular RNA (TRIzol Plus RNA Purification kit, Invitrogen)

This procedure uses TRIzol reagent to lyse cells, addition of chloroform to obtain an aqueous phase that contains RNA and a silica spin column to bind and elute RNA. Note: Wear gloves, lab coat and eye protection. The TRIzol contains phenol and guanidine isothiocyanate, and the samples should be handled in the Plexiglas hood until the RNA-containing aqueous phase is transferred to an RNA spin cartridge.

• Lyse adherent cells by adding 1 ml TRIzol /per 10 cm² (i.e. 6 well cluster). Use 200 µl for 24-well plate and 100 µl for 48-well plate. When treating cell suspensions, spin and discard culture medium, and add 1 ml TRIzol per 5-10 E6 cells. In either case, lyse cells by pipetting up and down. Transfer cell lysate to an Eppendorf tube, and stand 5 minutes at room temperature.
• Add 0.2 ml chloroform per 1 ml TRIzol reagent; cap securely and shake vigorously by hand for 15 seconds. (Too vigorous shaking, e.g. vortexing, may introduce DNA contamination into the RNA.) Stand 2-3 minutes at room temperature.
• Centrifuge 12K x g for 15 minutes at 4°C. (The mixture should separate into a lower red phenol-chloroform layer and a colorless upper aqueous layer containing RNA, ~600 µl... or 50% of the original 1.2 ml).
• Transfer the top (RNA) layer to a fresh tube and add an equal volume of 70% ethanol to obtain a final concentration of 35%; mix well by vortexing. Continue to mix as required to disperse any visible precipitate.
• Transfer the RNA to a spin cartridge (up to 700 µl) and spin 12K x g for 15 seconds at room temperature. Discard the flow-through.
• Add 700 µl wash buffer I to the RNA spin cartridge and spin 12K x g for 15 seconds at room temperature. Discard the collection tube.
• Place the RNA spin cartridge in a fresh wash tube, and add 500 µl wash buffer II (containing ethanol), and spin 12K x g for 15 seconds at room temperature. Discard the flow-through.
• Centrifuge the empty RNA spin cartridge 12K x g for 1 minute at room temperature to remove any remaining liquid.
• Add 500 µl wash buffer II and spin 12K x g for 15 seconds; discard flow-through, and then spin empty cartridge 12K x g for 1 minute at room temperature.
• Place the RNA spin cartridge in a clean RNA recovery tube.
• Add 30-100 µl RNase-free water to the center of the cartridge membrane, and stand at room temperature for 1 minute.
• Centrifuge the spin cartridge for 2 minutes at 12K x g at room temperature. Discard the spin cartridge.
• Determine the concentration and quality of the recovered RNA (OD 260/280) and store at -20°C.

• Perform qRT-PCR

RNA message levels are determined by qRT-PCR using commercially available reagent kits and supplies. Two Applied Biosystems 7900 Fast Real-Time PCR systems are used configured with the standard or the fast sample block respectively. In either case the 96-well reaction plates are sealed with optical adhesive covers and loaded into the cycler using a standard compression pad. Generally one-step kits are used, e.g. AB TaqMan RNA-to-C_T 1-Step, or Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System, using either TaqMan Gene Expression Assay components or LUX Assays (Light Upon eXtension primers, Invitrogen). Where appropriate, one-step SYBR green reagents are used with unlabeled primers. All assays contain ROX reference dye (where possible), and relative quantitation of target genes vs reference (calibrator) gene is determined (ΔΔC_T)

• Prepare Cell Lysates for Western Blot Analysis

To prepare cell lysates transfer 1E5 cells to a 2 ml Sarstedt (or 1.5 ml Eppendorf) tube; spin 5 minutes at 1600 rpm in RT6000 centrifuge (400 x G). Remove supernatant and resuspend in 50 µl distilled water. Immediately add 50 µl 2x sample buffer (for SDS Gels) and heat for 10 minutes at 100°C. 10 µl of lysate is generally appropriate for a 2 x 1.4 mm sample well. Laemmli SDS-PAGE and Western blot transfer is performed using standard procedures.

Cell Lines, Media and Reagents:

Maintenance of TLA-HEK293T Cells (Open Biosystems)

Medium:

DMEM (high glucose)

10% FBS (heated)

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

Notes:

1. The TLA-HEK293 cells do not need to be maintained in selection medium.
2. Transfection should be performed using medium without serum or antibiotics.
3. Working without antibiotics is a good idea and can be successfully done without infection using careful technique.

Starting cells from frozen stock-

• Remove cells from liquid nitrogen storage and quick-thaw in 37°C water, removing the vial just as the last ice crystals melt.
• Immediately dilute the thawed cells into cDMEM (with 10% FBS and L-glutamine) in a 15 ml conical tube.
• Spin 1000 rpm for 3-5 minutes at room temperature (~20°C)
• Carefully pour off the supernatant medium and resuspend the cells in 15 ml cDMDM, and transfer to a T25 flask or 100 mm dish.
• Place in the CO₂ incubator at 37°C.
• Fresh medium should be added every three days as indicated by medium color. Gentle treatment is required as the cells are easily dislodged from the flask surface.

Sub-culturing/Passaging the Cells

• The cells are passaged when 90% confluent and can be split using a 1:15 to 1:20 ratio. This will permit the cells to go for a number of days before reaching confluency, thus minimizing the amount of work required to maintain the cell line. Smaller passage ratios (e.g. 1:5) should be used if large numbers of cells are needed within a few days.
• Carefully remove the growth medium (by aspiration) followed by one wash with DPBS to remove medium components
• Add 0.05% trypsin solution (Gibco 25300) 2-4 ml per T150 flask (or proportional t the growth surface area of the flask or dish). Rock the flask gently to coat the surface, and then aspirate the excess, leaving a thin film of trypsin on the cells. The flask may be returned to the incubator for ~2 minutes to detach the cells.
• Add cDMDM to the flask using a 10 ml pipet, and pipet up and down ~5 times to produce a single cell suspension.
• Dilute the cell suspension into flasks or dishes as needed, and return to the CO₂ incubator.

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)-

Peptone 20g/l

Yeast extract 10 g/l

NaCl 5 g/l

Glycerol 8%

(Glycerol is added for long-term storage cultures but can be omitted in plasmid prep cultures.)

Note: This medium is modified from LB Broth Base (LB Broth, Lennox). The InVitrogen product #12780-052 contains 10g Select Peptone 140 and 5g Select Yeast Extract plut 5g NaCl per liter.

The above medium can be formulated using separate components; or, the Invitrogen LB Broth base can be used (20g/liter, which contains 10g peptone, 5g yeast extract and 5g NaCl) by supplementation with an additional 10g peptone (Fisher Tryptone BP1421-500) and 5g yeast extract (EMD 1.03753.0500).

2X-LB (Lennox)

InVitrogen LB Broth Base 10g

Fisher Tryptone 5g

EMD Yeast Extract 2.5g

d.i. water to bring volume to 420 ml

Microwave until just starting to boil; then autoclave 121°C, 15 psi, 15 minutes.

When cool, add either sterile d.i. water or 50% glycerol 80ml to 420ml 2X-LB, 16ml to 84ml, 8 ml to 42 ml, etc as needed. The glycerol is for preparation of frozen stocks while the 2X-LB broth without glycerol is for plasmid preparation, etc.

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.

Broth

• Weigh 10 g of LB broth base (Invitrogen #12780-052) into a 1 liter Pyrex bottle
• Add d.i. water to 500 ml
• Microwave and mix until dissolved.
• Check pH and adjust to pH 7.5 as needed.
• Autoclave 15 psi, 121°C for 15 minutes.
• Cool to room temperature.
• Aliquot into tubes/flasks for growth and add antibiotics for selection
• Zeocin stock is 100 mg/ml; dilute 1/4000 for 25 mg/ml final
• e.g. 6.25 µl per 25 ml medium in shake flask
• Carbenicillin stock is 10 mg/ml; dilute 1/100 for 100 µg/ml final.
• e.g. 250 µl per 25 ml medium

Agar plates

• In a 200 ml Pyrex bottle, add 2 grams LB broth base (Invitrogen),
• Add d.i. water to 100 ml
• Check/adjust pH and add 1.5 g agar (Bacto 214010).
• Microwave and mix until agar is melted and dissolved.
• Autoclave 15 psi, 121°C for 15 minutes.
• Cool to 55°C and add selective antibiotics-
• Zeocin (12.5 µl of 100 mg/ml stock per 100 ml agar/medium)
• Carbenicillin (0.5 ml of 10 mg/ml stock)
• Mix and pour ~20 into 100 x 15 mm Petri dishes (Fisherbrand 08-757-12).

(When fewer selective plates are needed, transfer the required amount of agar/broth to a sterile 50 ml conical tube before adding antibiotics. Use the remaining agar/broth to pour standard LB plates. e.g. 6.25 µl Zeocin and 250 µl Carbenicillin.)

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-

• Pick a single colony from a freshly streaked selective plate (e.g. with Zeocin & Carbenicillin, 25 mg/ml each) and streak second selective plate.
• Pick a single colony from the second plate, and inoculate 2 ml LB broth (with 25 µg ea Zeocin and carbenicillin); incubate at 35°C 300 rpm for 8 hr.
• Transfer 50 µl of the inoculum culture to 25 ml LBzc broth (containing carbenicillin) in a 125 ml baffled shake flask, and incubate 25°C 300 rpm for 12-16 hr.

Prepare reagents-

• Add Lyse Blue dye (1000x stock) to Buffer P1 (Tris-HCl, EDTA, pH 8) as needed, and add RNAse (100 mg/ml, 1000x stock for a final concentration of 100 µg/ml). Prepare 4 ml for each plasmid (midi) prep.
• Pre-chill neutralization buffer P3 (potassium acetate, pH 5.5).
• Warm buffer P2 (1% SDS + 0.2M NaOH) if precipitate is present.

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

• Transfer the bacterial cultures to a 50 ml polypropylene tube (Nalgene Oak Ridge 3119-0050) and centrifuge in Sorvall RC5 using the SA600 rotor at 8,000 rpm (6k x g) 15 min 4°C. Discard supernatant. (Pellets may be stored at -20°C pending further processing.)
• Mixed the chilled Buffer P1 (Tris-HCl, EDTA, pH8 with RNase A) so that the LyseBlue is evenly suspended, and transfer 4 ml to each pellet. Resuspend the pellet(s) by vortexing or pipetting up and down until all clumps are dispersed.
• Add 4 ml of buffer P2 (SDS/NaOH), and mix by vigorous inversion of the sealed tube 4-6 times; stand at room temperature (15-25°C) for 5 minutes. (Do not vortex or leave more than 5 minutes as this will promote shearing of the genomic DNA.) Cap buffer P2 immediately to avoid adsorption of CO₂. The solution should be a homogeneous blue color at this point.
• Add 4 ml of chilled buffer P3 (potassium acetate, pH 5.5) and mix immediately by inverting 4-6 times; incubate on ice for 15 minutes. Note, the viscosity of the solution should decrease as the genomic DNA, protein, cell debris and potassium dodecyl sulfate (KDS) are precipitated. Every trace of the LyseBlue should disappear.
• Centrifuge 13,000 rpm (20k x g) 30 minutes 4°C in the SA600 rotor. Remove the supernatant (containing the plasmid DNA) promptly, and transfer to a fresh tube.
• Centrifuge 13,000 rpm for 15 minutes 4°C, and remove the supernatant promptly. Save a 240 µl aliquot for gel analysis.
• Equilibrate a Qiagen-tip 100 by applying 4 ml buffer QBT (0.75 M NaCl, MOPS, pH 7, 15% IPA) and allow to empty by gravity flow.
• Apply the plasmid-containing supernatant from the second spin to a Qiagen-tip and allow to enter by gravity flow. Save 240 µl of flow-through for gel analysis.
• Wash the Qiagen-tip with 2 x 10 ml buffer QC (1M NaCl, MOPS, pH 7, 15% IPA). Save 400 µl of the combined wash fractions for gel analysis.
• Elute DNA using 5 ml buffer QF (1.25 M NaCl, Tris-HCl, pH 8.5, 15% IPA). Collect the eluate in a 15 or 50 ml polypropylene tube. Save 100 µl eluate for gel analysis.
• Add 3.5 ml (i.e. 0.7 volumes) room temperature isopropanol (IPA) to the eluted DNA. Mix and centrifuge immediately at 13,000 rpm, 4°C. Carefully remove the supernatant.
• Wash the pellet with 2 ml room-temperature 70% ethanol, and centrifuge at 13,000 rpm at 4°C.
• Air-dry the pellets for 5-10 minutes, and redissolve the DNAin TE buffer, pH 8.0 or 10 mM Tris-Cl, pH 8.5

DNA Quantification-

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

• Prepare a dilution of DNA in Tris-Cl, pH 7 (aim for 5-50 µg/ml). The Eppendorf UVette disposable cuvette can be used with as little as 50 µl, though 100 µl may be preferable.)
• µg DNA/ml = OD₂₆₀ x 50 x dilution

Agarose Gel Electrophoresis-

Gel preparation-

• Prepare TAE (tris-acetate EDTA) buffer
• 50x: 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA, pH 8 (final volume of 1 liter brought up with distilled water).
• 1x: 20 ml 50x up to 1 liter with d.w.
• Ethidiuim bromide stock, 10 mg/ml in d.w. (store at 4°C in light-proof bottle).
• 6x Gel loading buffer: In a 15 ml conical tube, add 25 mg bromphenol blue, 25 mg xylene cyanol FF and 4 g sucrose (or 1.5 g Ficoll 400) and dissolve in d.w. to a final volume of 10 ml. Store at 4°C (or RT for ficoll-containing buffer).
• Prepare sufficient agarose (e.g. Invitrogen UltraPure 15510-027)
• 50 ml required for small gel box (8 x 10 cm appx). Use 0.8% agarose for 11.7 kb plasmid.
• Weigh 0.4 g agarose into a 200 ml beaker; suspend in 50 ml 1x TAE; mark level, and cover loosely in plastic wrap. Microwave until agarose is melted/dissolved, and the solution is just beginning to boil.
• Cool on the bench (or in a water bath) to 55-60°C. Replenish any volume that may have been lost by addition of d.i. water, and add 2.5 µl of 10 mg/ml ethidium bromide stock.
• Place a glass plate inside the gel box and place the comb 1 cm from one end of the box, allowing 0.5-1 mm clearance from the base and pour the agarose solution into the gel box to a depth of ~3-5 mm.
• When the agarose is solidified, add a small amount of TAE buffer to the surface of the gel, and remove the comb. Using a pipet tip, push up on the glass bottom, and transfer the glass slide with agarose gel to the running box. Add sufficient TAE to fill the electrode chambers and to cover the gel with 1 mm depth buffer. Ethidium bromide may also be added to the TAE running buffer (to 0.5 mg/ml).
• Prepare samples- 0.2-0.5 µg of plasmid DNA can be loaded per lane. (As little as 20 ng may be detectable.)
• In an Eppendorf tube add DNA and 1x TAE (10 µl combined volume) followed by addition of 2 ul gel loading buffer.
• Rinse the sample wells with 1X TAE, and apply the samples
• Connect electrodes so that the anode is on the opposite end of the gel box from the sample wells. Turn on the power supply, applying 1-10 V/cm until the tracking dyes indicate adequate separation. (Running voltage for the small gel box would be 18-180 V. Starting with 50-100 volts should be adequate.)