Protocols

Recipes:

Antibiotics for bacterial cloning: 

Ampicillin (sodium salt): 500x stock solution: 50 mg/ml in water (store at -20ºC); working concentration is 100μg/ml

Carbenicillin: 1000x stock solution: 50 mg/ml in water (store at -20ºC); working concentration is 50μg/ml

Kanamycin: 200x stock solution: 10 mg/ml in water (store at -20ºC); working concentration is 50μg/ml

Chloramphenicol: 200x stock solution: 34 mg/ml in ethanol (store at -20ºC); working concentration is 170μg/ml

Tetracyclin HCl: 100x stock solution: 5 mg/ml in ethanol(store at -20ºC); working concentration is 50μg/ml

Streptomycin: 200x stock solution: 10 mg/ml in water (store at -20ºC); working concentration is 50μg/ml

Cell Freezing Medium: Consists of 75% base medium, 10% DMSO, 15% FBS.

Collagenase IV: To make a 100x (10% w/v) concentrated stock, dissolve 1g collagenase powder in sterile D-PBS, q.v. 10ml. Gently rock the solution back and forth until the the collagenase is dissolved. Filter sterilize using a 0.2μm syringe filter. Aliquot into sterile screw top microcentrifuge tubes. Store at -20ºC. Use a final concentration of 0.1% w/v Collagenase when digesting breast tissue.

DNA Loading Buffer(Ficoll): 15%  w/v Ficoll (type 400) polymer in distilled water; 0.25% w/v Bromphenol Blue (optional: 0.25% w/v Xylene Cyanol FF).

Drugs for Cell Selection (prepare stock solutions in fume hood):

Puromycin: (Sigma, P8833-25MG):  Soluble in water (50 mg/ml)

Hygromycin: (Sigma, H3274-50MG): Hygromycin B is soluble in water, at concentrations >50 mg/mL, and stable in aqueous solution for at least five years at 2-8°C.

Blasticidin: (Thermo, R21001):  Use 2–10 µg/mL Blasticidin for selection in mammalian cells. Prepare Blasticidin stock solutions of 5–10 mg/mL by dissolving Blasticidin in sterile water and filter sterilize the solution. Blasticidin is soluble in water and acetic acid.  Aqueous stock solutions are stable for 1–2 weeks at 4°C and 6–8 weeks at −20°C. Medium containing Blasticidin can be stored at 4°C for up to 2 weeks. pH of the aqueous solution should not exceed 7.0 to prevent inactivation.

ER Tracker Green:  “ER-Tracker™ Green dyes  are supplied as 100ug of lyophilized material. Prepare a 1 mM stock solution by dissolving the contents of the vial in 128uL of DMSO; Make aliquots of this 1 mM (1000x) solution and store frozen with desiccant. To minimize potential labeling artifacts, use the lowest dye concentrations possible. Best results are obtained when staining is performed in Hankʼs Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg, Gibco cat. #14025-092) at 37°C/5% CO2.  For adherent cells, remove the medium from the culture dish, rinse with HBSS, and add prewarmed staining solution. Incubate the cells for approximately 15–30 minutes at 37°C. Replace the staining solution with fresh probe-free medium and view the cells using a fluorescence microscope.” -Life Technologies product sheet

2.5M Glycine: To make 1Liter: Dissolve 75g Glycine (Sigma G7126-5KG) into  900ml distilled water; then q.v. 1L.  Filter Sterilize and store at room temp.

Image-iT Green Caspase detection: PDF

Immunofluorescence staining buffer:  To 80ml of 1x PBS, add 10ml 5 % Saponin solution and 10ml goat serum. Filter sterilize through 0.2 μm filter. Store at 4 deg. C.

MitoTracker Red CMXRos: To prepare a stock solution: Allow the product to warm to room temperature (it is stored at -20). Dissolve 50ug lyophilized dye (contained in each tube) with 94ul of high-quality anhydrous DMSO to achieve a 1mM stock solution. (The MW of MitoTrackerR CMXRos is: 531.52). Make aliquots of this 1 mM (1000x) solution and store frozen, protected from light. The concentration of probe for optimal staining varies by application. General Guidelines: Use working concentrations of 25–500 nM. For staining cells that are to be fixed and permeabilized (see Fixation and Permeabilization after Staining), use a working concentration of 100–500 nM. Grow cells on coverslips, add prewarmed staining solution containing the mito-tracker probe and incubate 15-45 minutes. Refresh with pre-warmed medium and image.  To Fix: carefully remove the medium/buffer covering the cells, and replace it with freshly prepared, pre-warmed buffer or growth medium containing 2–4% formaldehyde.  -Life Technologies product sheet

Normocin:  is supplied as a 500x concentrate. We carry freshly sorted cells in 0.5x. So, to a new 500ml bottles of medium, add 500ul of Normocin. Indicate it has been added by marking a ‘+N’ on the bottle!

PBS/A: Phosphate Buffered Saline with Antibiotic/Antimycotic is used to rinse fresh tissues prior to digestion with collagenase. To make, add 10 ml 100x penicillin/streptomycin and 10 ml 100x Fungizone to 500 ml PBS. Mix and store at 4ºC (the final concentration is supposed to be 2x).

Polyhema & coating dishes: To make a 20mg/ml solution, dissolve 2g Polyhema (Poly(2-hydroxyethyl methacrylate); Sigma # P3932-10G) in 100ml 95% EtOH. Heat to 65ºC x 3 hours. Cool and store at room temp.  To coat a dish with polyhema, add the appropriate* amount of the20mg/ml solution to the dish (or each well of a multi-well dish) and let dry overnight at 37ºC.  Coated plates can be wrapped and stored at room temperature until ready for use.       *use ~70 μl polyhema per cm2 growth area; e.g., 5.25 ml per T75; 4 ml per 100mm dish, etc.

Primer dilutions: Dissolve lyophilized primers in TE to a 100uM final concentration. Warm to 37°C & mix; store at -20ºC.  Primer mixes: nearly all primer mixes we use are 3uM FOR/3uM REV (10x concentrated) , diluted in PCR grade water.  For example, to make 200ul of a mixture containing forward and reverse primers, add 6ul FOR primer and 6ul REV primer to 188ul PCR grade water. Store at -20°C.

Saponin Stock Solution (10x, 5% w/v):  To create a 5% w/v solution (10x), dissolve 5 grams of Saponin (Sigma 47036-50g-F) into 80ml water. Mix thoroughly until dissolved by rocking overnight at 4 deg C. q.v. 100ml, filter sterilize through 0.2 um filter, and store at 4 deg C.

Trypsin Inhibitor:  Remove 20ml of D-PBS (without Ca++ or Mg++) from a new 500ml bottle. Use it to dissolve 500 mg of Trypsin inhibitor from Glycine max (soybean, Sigma T9128-500MG). Filter sterilize through 0.2 μm syringe filter. Add back to the 480 ml D-PBS and mix.  The final concentration is 1 mg/mL , or 0.1% w/v.

 

Brief Protocols:

Collagen I coating of dishes:  PureCol (Bovine Collagen I, Advanced Biomatrix) is supplied at ~3mg/ml.  Dilute to 1/46 with D-PBS to 65 ug/ml, mix,  and apply enough to cover the surface of the culture vessel.  Incubate for at least 30 min at room temp (or long-term at 4ºC).

Phalloidin Staining: Wash cells with PBS. Fix with 4%PFA x 10 min. DO NOT USE METHANOL- it will disrupt actin filaments (use methanol-free formaldehyde). Treat cells with 0.1% Triton-X 100 (or -20° Acetone -if using glass) for 3-5 min. Wash 2x with 2% PBS. Optional: block with 2% FBS (in PBS) for 15-30 min. Dilute 5ul of methanolic stock of phalloidin by adding it to 200ul 2% FBS and add it to the section/cells. Incubate for 20min at room temp (4°-37° is acceptable). Wash with PBS. Image immediately. For long term storage, air dry

Subculturing (splitting) primary cell cultures: Aspirate growth medium and wash the cells with PBS(Ca++/Mg++ free). Apply enough ‘Cell Dissociation Reagent’ (CDR; Sigma, #C1419) to cover the cells. Each cell type has a different degree of attachment to the dish and to adhesiveness to each other, so the required time in CDR varies and is best determined empirically. Incubation times typically vary between 5-10 min (at room temp). While some cell types will have lifted off the plate, most will not and will need to be treated with trypsin(also at room temp). The amount of trypsin and time will also need to be empiracally determined, so monitor closely by phase microscopy. The most common concentrations of trypsin (0.25% or 0.05%) are both ok, but will obvioiusly alther the amount/times needed to get the cells fully dissociated. A gentle tapping of the dish or pipetting may be necessary to fully suspend the cells. Once the cells are off and not in clumps (they should be single cells), stop the trypsin by adding at least an equal volume of 0.1% w/v Soybean Trypsin inhibitor.  Each cell type will appear differently at this stage and there can be lots of additional debris. Filter the cells through a 40um filter if they are to be FACS sorted.  Count the cells and record their suspended volume. Place the cells in a 15ml conical tube, dilute with PBS (fill to 10-15ml mark), and pellet the cells by centrifugation (100-200g is sufficient for cultured cells). The cells are now ready to be archived (resuspend in cell freezing medium) or placed back into culture (normal growth medium).

 

 

Detailed Protocols: 

Tissue immunostaining (cryosections):  PDF

Cell immunostaining:  PDF

Collagenase digestion of breast tissues:  PDF

Dissociating organiods and preparing cells for FACS: Add link here

Immunolabeling cells for FACS analysis: Add link here

Tri-lineage differentiation of mesenchymal stem cells:  Add link here

TOPO Cloning for ‘Multisite Gateway’ (5’TOPO & D-TOPO):  Add link here

LR recombination reactions for ‘Multisite Gateway’ (5’TOPO & D-TOPO):  Add link here

Lentiviral Methods (packaging and titrating virus):  PDF

Lentiviral Packaging Excel Worksheet (for calculating plasmid volumes):  Excel Sheet

Cell Incubator Care and Cleaning:  PDF or Thermo Website

 

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