PROTOCOLS FOR CELL PLATING AND                  shRNA / LENTIVIRAL PREPARATION RESULTS

Download the Plating Protocol PDF

Cell Plating and Long Term Growth Instructions

Simplified Protocol for Plating and Culturing Primary Neurons (Hippocampal and Cortical)

Time required: 2 - 4 hours Preparation time: 1 day prior to use

3 steps for culturing neurons: coat (1), thaw (2) and plate (3):

Solutions required

MEM/FGH Medium - plating medium: MEM with 10% FBS, glucose and HEPES

NB27G - maintenance medium: Neurobasal with B27 and GlutaMAX

(aliquots of MEM/FGH and NB27G medium to be used with cells must be temperature/pH prebalanced by placing in a tissue culture dish for at least 1hr in the incubator)

PL Solution - coating solution (PL): poly-L-Lysine 0.1 mg/ml in borate buffer

Coating (dishes or coverslips)

  1. If you are using glass bottomed culture chambers, regular multi-well plate or cell culture dishes,coat them with PL solution overnight.

  2. The next morning, wash with sterile water 3X and fill with MEM/FGH medium, and balance in CO2 incubator for at least 2 hours before plating.

  3. If you are using coverslips for culturing, first place coverslips in 70% nitric acid overnight, wash 8-10X with excess volume (at least 10X volume of acid added) milliQ purified water, and bake them in oven at 225degC for 4 hours or longer (alternatively, if using coverslip holding racks, UV sterilize in tissue culture hood with blower on for several hours).

  4. Coat coverslips as described in steps 1 & 2.

Thaw

  1. Prebalance several mL of MEM/FGH medium in CO2 incubator for 30 minutes prior to thawing cells in tissue culture dish.

  2. If you are planning on thawing multiple vials, perform the following steps one vial at a time.

  3. Take a vial, put it in 37degC water bath and take it out when almost all ice is thawed, but do not keep the vial in the water bath for more than 2 minutes.

  4. Spray the outside of the vial with 70% ethanol and wipe with paper towel. Quickly proceed to the plating step for each vial.

Plate*

  1. Slowly pipette the thawed cell solution with 1ml tip to pre-coated plates/dishes/coverslips (depending on your application and cell density), avoiding any rapid pipetting and tips with small openings (wide bore at least 1.5mm). Use plating chart below for guidelines.

  2. Take 1 ml of MEM/FGH medium and place back in vial to wash any neurons that remain at the bottom and on tube walls. Pipette out the 1mL to the plates/dishes/coverslips equally.

  3. Swirl the plates/dishes/coverslips slowly for 20 seconds by hand rotation, in opposing directions, to equally distribute the neurons and return to CO2 incubator.

  4. Repeat thaw and plate steps for all vials.

  5. Neurons should start attaching within 30 minutes after plating. After 2-4 hours, replace MEM/FGH medium with pre-balanced NB27G medium.

  6. After 3-4 days, remove 1/3 of old NB27G medium from neurons and replace with an equal volume of fresh, pre-balanced (in CO2 incubator) NB27G medium (can add 1.25uM cytosine arabinose final concentration of total medium; use stock 3.75uM in NB27G). Repeat this procedure on day 7, 14 and 21, without cytosine arabinose addition.

*Note - See plating chart below for guidelines for optimal neuron morphology. Plating neurons at higher density will yield more robust cultures over time. To ensure healthy cultures, neurons should not be plated at a density lower than 300K per 60mm dish, 100K per well of 6-well dish or 35mm dish, or 50K per 12-well dish.

Media/Solutions composition

1. MEM/FGH Medium – MEM (with Earles salts and sodium pyruvate, no glutamine) with 10% FBS, 33mM glucose and 10mM HEPES. Adjust pH to 7.3 and sterile filter.

2. NB27G - maintainence medium: Neurobasal (Life Technologies) with B27 (Life Technologies) and GlutaMAX (Life Technologies). Okay to substitute B27 with GEM21 NeuroPlex from Gemini Bio Products.

3. PL solution: Make up 0.1mg/mL poly-L- or poly-D-lysine from 1.0mg/mL stock dissolved in borate buffer (dissolve 0.72g borax (sodium tetraborate) and 0.81g boric acid in 100ml of water, pH to 8.5) and sterile filter. This must be made up fresh, and plates not allowed to dry after PL solution coating.

**Note - cell numbers for immunofluorescence/transfections are for low density plating, and should be scaled up when troubleshooting cell health**

 

shRNA and CRISPR Transduction Technology

We provide custom services that are in-house prepared using our acquired expertise that our clients have benefitted from for years. Our service includes custom shRNA construct preparation targeting any gene of interest. 

shRNA constructs are comprised of several options for client choice:

  • selection markers (fluorescent GFP or other color, antibiotic resistance)
  • inducible or constitutive promoter (U6 or H1)
Percentage knockdown of 36 shRNA constructs targeting 6 genes (including control). On average >70% of our designed constructs transduce at a 70% or greater expression and transcript knockdown as determined by Q-PCR.  

Percentage knockdown of 36 shRNA constructs targeting 6 genes (including control). On average >70% of our designed constructs transduce at a 70% or greater expression and transcript knockdown as determined by Q-PCR.  

We provide several services for construct design. We will design your target gene of interest in a standard shRNA vector or clients can choose a custom design based on their needs. All target gene constructs are cloned and verified before packaging into LentiPrep lentivirus particles. Validation of construct knockdown is also available to ensure rapid, guaranteed effect. However transcript knockdown does not always correlate with phenotypic effect (our technical scientists can guide clients through this selection process).

Similar services are provided for shRNA and CRISPR knockdown approaches. Data packets for each are available on request. 

(A) HEK293 cells (DIC) were transduced with LentiPrep particles expressing lacZ gene, and co-transduced with either LentiPrep particles containing shRNA to lacZ (lacZ), negative control lamin (lamin) gene, or mock transduced (Mock), with all vectors containing a co-expressed GFP marker (GFP) on the plasmid for transduction confirmation. (B) B-galactosidase activity assay to determine extent of shRNA knockdown of lacZ expression in cells from panel (A). B-galactosidase activity was normalized to control cells containing lacZ transduction alone, while shRNA co-transduced cells showed decreased lacZ expression depending on the gene targeted. This is in contrast to control cells with no lacZ expression which show no significant level of activity (n=3-5 experiments per treatment). (C) Western blot for lamin (shRNA knockdown) or actin (control, empty shRNA vector) proteins after stable transduction and expression of shRNA LentiPrep contructs. Each cell population (#1-5, bottom) were tested for lamin (lam) and lacZ (lac) expression, and all showed significant knockdown of lamin proteins (antibody recognizes lamins A/C as two separate sized bands at 68-78kD). Marker (M) shown on left side with sized bands.

(A) HEK293 cells (DIC) were transduced with LentiPrep particles expressing lacZ gene, and co-transduced with either LentiPrep particles containing shRNA to lacZ (lacZ), negative control lamin (lamin) gene, or mock transduced (Mock), with all vectors containing a co-expressed GFP marker (GFP) on the plasmid for transduction confirmation. (B) B-galactosidase activity assay to determine extent of shRNA knockdown of lacZ expression in cells from panel (A). B-galactosidase activity was normalized to control cells containing lacZ transduction alone, while shRNA co-transduced cells showed decreased lacZ expression depending on the gene targeted. This is in contrast to control cells with no lacZ expression which show no significant level of activity (n=3-5 experiments per treatment). (C) Western blot for lamin (shRNA knockdown) or actin (control, empty shRNA vector) proteins after stable transduction and expression of shRNA LentiPrep contructs. Each cell population (#1-5, bottom) were tested for lamin (lam) and lacZ (lac) expression, and all showed significant knockdown of lamin proteins (antibody recognizes lamins A/C as two separate sized bands at 68-78kD). Marker (M) shown on left side with sized bands.

Lentiviral Transduction in Primary Neurons

LentiPrep targeted shRNA against Tau can efficiently knockdown Tau protein (red) after 72 hours. DAPI stained nuclei (blue) identify cell numbers after 6 DIV.

LentiPrep targeted shRNA against Tau can efficiently knockdown Tau protein (red) after 72 hours. DAPI stained nuclei (blue) identify cell numbers after 6 DIV.