Separation of normal CD34+ cells from fresh pheresis of mobilized stem cells

1.      Resuspend sample up to 100 mL of MACS buffer (see recipe below). Aliquot to two 50-mL conical tubes. Centrifuge for 10 minutes at 1000 rpm to "soft spin" the pellet. A soft spin keeps the platelets, which are concentrated in pheresis samples, in the supernatant. Platelets cause major problems with the staining of the sample as well as the running of the sample through the magnetic column. The supernantant must be aspirated, not poured off, since the pellet is loose.

2.      If the supernatant from the soft spin is still relatively cloudy, the soft spin may be repeated.

3.      ACD-A changes the density of the cells in the pheresis sample so that granulocytes will stay at the interphase of the Ficoll. Although pheresis samples have a high concentration of granulocytes, it is more important to remove the platelets before they activate. An alternative protocol may be that the sample is initially suspended in buffer without ACD-A and then Ficolled. The mononuclear layer may then be resuspended in buffer with ACD-A, and the soft spin performed. We have found it best to just leave the granulocytes and adjust concentration of the antibody (follows).

4.      Count the total number of cells. Combine sample into one 50-mL conical tube. Miltenyi lists antibody amount according to the total number of cells; however, this may be adjusted according to the estimate of number of cells positive for the sorting parameter. Since pheresis has between 1-10% CD34+ cells, we usually use 50% of the recommended amount of antibody. If a Ficoll is not performed, this may be reduced to ? the amount of antibody, but the buffer should not go below ?, and the incubation time should be extended to 30 minutes. Add ? the amount of buffer recommended by Miltenyi. Add ? the amount of reagent A1, shake gently. Add ? the recommended amount of reagent A2, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.

5.      Wash the sample two times with 50 mL of MACS buffer.

6.      Resuspend the sample in _ the recommended amount of buffer, and _ the amount of reagent B, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.

7.      Wash the sample one time with 50 mL of MACS buffer.

8.      Resuspend the sample in at least 10 mL degassed (see Note) MACS buffer for 1 x 109 total cells, or up to 20 mL for 2 x 109 total cells. Run sample over a VS positive selection column.

9.      Wash 2X with 3 mL of degassed buffer.

10.   Attach stop cock and syringe to bottom of VS column. Remove column from magnet. Backflush column with 6 mL of degassed buffer. Replace column in magnet.

11.   Remove stop cock. Allow buffer to flow through column. Wash 2X with 3 mL of buffer.

12.   Remove column from magnet. Add 6 mL buffer to column and allow to run through. Add 6 mL buffer to column and plunge the column.

13.   Count the total number of cells collected from each fraction to calculate the recovery of the separation.

14.   Perform flow cytometry on the collected fractions to assess sample purity with CD45-FITC and CD34-PE (Becton Dickinson).

MACS buffer

Hank's Balanced Saline Solution (HBSS) -Ca+2, -Mg+2

0.5% BSA

0.6% Anticoagulant Citrate Dextrose- Formula A (ACDA) (Baxter)

Filter sterilize and store at 4°C

Notes: It is very important when running the magnetic column that only degassed MACS buffer be used. To degas the buffer, place 100 mL of buffer in a 150-mL bottle. Place a rubber stopper attached to a vacuum line over the mouth of the bottle. Turn vacuum on. Allow the buffer to degas at room temperature for at least 30 minutes. Replace cap on bottle and refrigerate buffer until cold. Use buffer as directed.

Mouse Tumor Biology (MTB) Database

The Mouse Tumor Biology (MTB) Database supports the use of the mouse as a model system of hereditary cancer by providing electronic access to:

l  Information on endogenous spontaneous and induced tumors in mice, including tumor frequency & latency data,

l  Information on genetically defined mice (inbred, hybrid, mutant, and genetically engineered strains of mice) in which tumors arise,

l  Information on genetic factors associated with tumor susceptibility in mice and somatic genetic-mutations observed in the tumors,

l  Tumor pathology reports and images,

l  References, supporting MTB data

l  Links to other online resources for cancer

http://tumor.informatics.jax.org/mtbwi/index.do

IL-2 ELISPOT

Materials Required
·  Peptides of desired specificity (stock at 10mM in DMSO - may be aliquotted and stored at -20ºC)
·  Phytohemagglutinin (PHA; optional) for use as a control for cytokine production (Sigma #L8902)
·  Purified anti-IL-2 capture antibody (e.g. clone IL2-I, Mabtech #3440-3)
·  Biotin-conjugated anti-IL-2 detection antibody (e.g. clone IL2-II, Mabtech #3440-6)
·  Streptavidin-alkaline phosphatase 1mg/ml (e.g. Vector Labs SA-S100)
·  BCIP/NBT alkaline phosphatase kit (e.g. Vector Labs SK-5400)
·  100mM Tris -Hcl, pH9.5 for color development
·  35% ethanol (v/v in distilled water)
·  Sterile phosphate buffered saline (PBS)
·  Sterile distilled water
·  Complete R10 medium (RPMI-1640 supplemented with 10mM HEPES, 50mM 2-mercapto-ethanol, 100U/ml Penicillin,0.1mg/ml streptomycin, 2mM L-Glutamine and 10% fetal calf serum)
·  96 well PVDF membrane ELISPOT plates (Millipore #MSIPS4510)

Recommended Experimental Controls
·  Negative Control – no antigen stimulation / stimulation with known negative peptide
·  Positive Control – Stimulation with PHA

Protocol applicable for IL-2 ELISPOT assay
Aseptic Procedures (Use sterile buffers and aseptic conditions; use laminar flow hood for procedures)
1. Pre-wet plate wells with 15ml 35% ethanol for 1 minute. Aspirate, then wash twice with 200ml dH2O before the ethanol evaporates.
Once the membrane is pre-wet with alcohol, do not allow membrane to dry for the duration of the assay.
2. Dilute anti-IL-2 capture antibody to 15mg/ml in sterile PBS. Coat plate with 50ml/well. Incubate at 4°C overnight.
3. Decant or aspirate coating antibody from plate.
4. Wash plates 5 times with 300 ml/well sterile PBS. Decant.
5. Block plate with 200 ml/well of complete R10 medium at room temperature for at least 1 hour. Decant or aspirate plate.
6. Aliquot up to 300,000 cells/well in 100 ml complete R10. Aliquot peptide ([5mM] final) or controls diluted in complete R10 medium to appropriate wells.
The desired cell concentration depends on the intensity of the immune response. If the expected response is unknown, then we recommend a serial dilution of cell concentrations.
7. Incubate for 23-36 hours at 37°C, 5% CO2 and 95% humidity.

Incubation times should be developed and evaluated by the user. However, antigen-specific stimulation of cells results in detectable spots within 23 hours.

Non-Aseptic Procedures
8. Decant cells and medium from plates. Wash plate 5 times with 300ml PBS.
9. Dilute biotin-conjugated anti-Il-2 detection antibody in PBS and filter. Add 50ml/well to plate and incubate at room temperature for 3 hours.
Failure to filter the detection antibody may result in non-specific spot formation due to protein aggregates.
10. Decant antibody solution. Wash 5 times with 300ml PBS. Allow wells to soak for 1 minute for each wash.
11. Dilute alkaline-phosphatase reagent in PBS (final concentration 1mg/ml). Add 50 ml to each well and incubate at room temperature for 1 hour.
Exceeding 1 hour incubation with enzyme conjugate will result in increased background.
12. Discard solution. Wash plate 5 times with 300ml PBS.
13. Add NBT/BCIP reagent as per the manufacturers instructions and leave the substrate to develop until spots are clearly visible (approximately 30 minutes)
Optimization of substrate development time is critical, since over-development will result in increased background.
14. Stop the substrate development by washing wells 3 times with dH20.
15. Remove the under-drain from the plate and allow to air-dry.
16. Count spots by eye, or using an automated ELISPOT plate reader.

anti-HA antibody Western Blotting Protocol

1) Run gel (in 1:10 running/transfer buffer(10x) and H₂O for a total of 1litre) at 150 volts until leading bromophenol blue band is nearing the bottom edge of the glass plates or according to the expected migration of the protein of interest.

2) Transfer proteins in acrylamide gel to nylon membrane.

3) Block overnight @4℃ in PBS (or TBS, it doesn't matter) with 5% non-fat dry milk. I dilute 2.5 mg in 50 ml.

4) Wash off excess milk sol'n with PBS x3 .(important if you are re-using your antibody)

5) Incubate in primary antibody solution: I use PBS with 0.1% TWEEN-20 plus 5% BSA as the basic solution, but you can use TBS too. I dilute the HA antibody 1:5000, just to be sure you will see a signal. I haven't EVER seen appreciable background. I've also used it at 1:10K without any appreciable loss of signal. I usually go 30-90 minutes at RT (you can save the 1ab solution and use for a month or so).

6) Wash with 12-15 quick washes with PBS/Tween ( I literally pour it in, shake it around for 5-10 seconds, and pour it off: this method was worked out in the Yamamoto lab eons ago).

7) Incubate with secondary antibody (anti-mouse) in 5% BSA/PBS/Tween as you would ordinarily. I usually go for 2 hours @ room temp.

8) 12-15 quick washes again(in 1XPBS).

9) Assay for chemiluminescence or whatever you want. The normal procedure is to submerge blot in 20ml of ECL reagent for 2 minutes. Then wrap blot in saran wrap and wipe away excess ECLreagent. Place blot in film cartridge and bring to dark room. Expose film for ~2 mins.

Notes on the primary antibody:

Source : Covance/Babco

Description: Crude ascites fluid monoclonal anti-HA antibody (CATLN#135032002, 11mms-101R, 0.5 ml)

Handling: Aliquoted into 10ul portions in -20℃

Internal notes: labelled anti- Ha (Marc’s 1:5000)

Far Western Protocol

1. Run samples out on a gel. For bacterially expressed proteins, generally 5 μl is plenty (1ml cell cμlture; cells resuspeded in 50 μl loading buffer). Run the gels (BioRad mini gels) at 195 V for approximately 40 min. (until samples run close to the bottom of the gel).

2. Transfer the proteins from the gel onto nitrocellμlose. For the BioRad setup, the case shoμld be set up as follows: black side down, then 3M Scotch Brite Pad, then blotting paper, gel, nitrocellμlose, 2^nd blotting paper, 2^nd Scotch Brite Pad, and the clear side of the case. Put the case in the holder, black side of the case facing black side of the holder. Run the transfer at 100V for 1 hour.

3. Meanwhile, prepare 500 ml of AC Buffer (+ Tween):

50 ml glycerol (= 10% glycerol)

10 ml 5M NaCl (= 100mM NaCl)

10 ml1M Tris, pH 7.6 (= 20 mM Tris)

1 ml 0.5M EDTA (= 0.5mM EDTA)

5 ml 10% Tween-20 (= 0.1% Tween-20)

put on ice

b) Make 50 ml (or more) of 2% milk powder solution:

50 ml AC Buffer

1 g milk powder

-put on a rocker to dissolve the milk powder (may take 20-30 minutes). Then put on ice

4. Make the probe, using the TnT (Promega) Reticμlocyte Lysate kit.

Set up either a 25 μl reaction or a 50 μl reaction, depending on the size of tray you'll be using for washes and probing. Below is the recipe for a 50 μl reaction mix:

25 μl Reticμlocyte lysate (I use a little more, ~27 μl)

2 μl Reaction Buffer

1 μl T7 (or T3) polymerase (or other polymerase)

1 μl amino acids minus methionine (or missing other amino acid)

1 μl RNA Guard

4 μl 35S-met (or other labelled amino acid)

16 μl DNA + ddH₂O (1-2 μg DNA)

Spin down the sample (pμlse spin) to remove air bubbles. Let the reaction proceed at room temperature for 1 hour, 10 minutes (can be longer or shorter, depending on the protein).

Block, Probe, and Wash

5. After the transfer is complete, put the blot into a tray. Keep the side that was touching the gel up). Do 1-2 quick washes with 1X PBS to remove the SDS. Then, do a quick rinse in AC Buffer, to remove the PBS. Pour on the 2% milk powder (just enough to cover the blot), put a lid on the dish, and rock the blot at 4℃ for 1 hour. This is the blocking step.

6. Meanwhile, set up 2 spin columns:

Use 1cc syringes, remove the cap and the plunger. Put in glass wool to fill the opening at the bottom, push down with the plunger. Put the syringe in a 15 mL falcon tube. Add BioRad G 25 resin, stored at 4℃ in TE buffer, to the syringe , filling to the top (avoid air bubbles!). Spin down at 2000 rpm for 3 minutes. Then refill with G-25, and repeat the spin cycle. (At this point, the G-25 shoμld be packed down such that it occupies about 0.8ml.

Add to the columns 100 μl AC Buffer, spin at 2000 rpm for 3 min. Repeat two more times.

7. When the probe labeling reaction is finished, dilute the sample using AC Buffer to make a final volume of 100 μl. Load this on the spin column and spin at 2000 rpm for 3 minutes in a fresh falcon tube. Collect the flow-through. This process removes unincorporated nucleotides.

8. When there are approximately 20 minutes left in the blocking step, it is time to prepare the probe mix. At this point, you may want to collect a 2μl sample of your probe (from the 100 μl), and combine it with 10 μl 1X SDS gel loading buffer. You can then run this sample on a gel, and put film on the dried gel to see if the probe labelling step actually worked.

The volume of the probe mix depends on your initial probe reaction mix volume, and the size of tray that you are using. For example, if you set up a 50 μl initial reaction, you can use up to 20 ml of probe mix. For a 25 μl reaction, use no more than 10 ml of probe mix.

RNA isolation and quantification Protocol

The acid-phenol extraction is a simple procedure to extract RNA from cells, and to separate RNA from DNA. When cells are lysed and extracted with phenol, protein and lipids always partition into the phenol layer whereas RNA partitions to the aqueous layer (regardless of the pH of the phenol). In contrast, DNA shows a pH-dependent partitioning. When the aqueous phase of the phenol is buffered above pH 7.5, DNA partitions with RNA into the aqueous layer. However, when the aqueous phase of the phenol is buffered at pH 4.0, DNA will partition into the phenol layer. The concentration of RNA can be conveniently measured due to its ability to absorb light at 260 nm. The ratio A₂₆₀:A₂₈₀ is an indication of the purity of the RNA. The ratio (for pure RNA) should be 1.8 -2.1. However, even if the ratio is less than 1.8, the RNA quality may be acceptable and can be confirmed by the subsequent gel electrophoresis.

Materials required:
TRIzol Reagent (Gibco BRL; DNA and protein can also be extracted from the samples using this reagent)
Chloroform       Isopropanol        75% ethanol         RNase-free water (i.e. DEPC treated)
 

Phenol - Caution Phenol is highly toxic, even in small amounts. If spilt on skin - wash immediately under cold water If spilt on clothing - remove immediately If spilt on bench, mop up immediately and consult a demonstrator. UV light -Caution Spectrophotometers are a source of UV radiation

RNA isolation
1. Discard the cell growth medium safely.
2. Wash the cell monolayer by carefully by adding 1 ml PBS to the side of the dish making sure you do not disrupt the cells. Pour off the PBS wash.
3. Add 1 ml TRIzol to the plate to lyse the cells. Pass the cell lysate several times through a pipette (insufficient TRIzol may result in contamination of the isolated RNA with DNA).
4. Transfer the lysate to a 1.5 or 2.0ml Eppendorf tube.
5. Incubate the homogenized samples for 5 min at room temperature (this allows complete dissociation of nucleoprotein complexes).
6. Add 0.2 ml of chloroform per 1 ml of TRIzol (Make sure the tube is tightly capped).
7. Shake tubes vigorously by hand for 15 sec and incubate at room temp for 2.5 min.

RNA/DNA separation
1. Centrifuge samples at no more than 12,000 rpm for 15 min at 4℃. Following centrifugation the mixture separates into a lower, phenol-chloroform phase (red), an interface, and an aqueous upper phase (colourless) Ð RNA remains exclusively in the aqueous phase.
2. Transfer the aqueous phase (i.e. top layer; »600 µl) to a fresh tube.
3. To precipitate RNA, add 1 ml isopropanol per 1 ml of TRIzol used for the initial lysis and mix by inversion, then incubate at room temp for 10 min.
4. Centrifuge at 12,000 rpm for 10 min at 4ûC (cold-room)
5. Discard the supernatant and wash the RNA pellet with 75% ethanol (use at least 1 ml of 75% ethanol per 1 ml of TRIzol used for the initial lysis) - mix by vortexing.
6. Centrifuge at 7,500 rpm for 5 min at 4℃ (cold-room).
7. Discard the supernatant and (briefly) air-dry the RNA pellet by inverting the Eppendorf tube for 5-10 min. It is important NOT to dry the RNA completely.
8. Dissolve RNA in about 20ml RNase-free water
9. Heat RNA at 55 ûC for 5-10 min (increases solubility of RNA). Store at -20℃ o/n.

RNA concentration measurement
1. Dilute 2 - 3 µl of RNA (e.g. approx. 1/10 of RNA from above) into 500µl H₂O.
2. Place in a quartz cuvette and measure the OD₂₆₀ and OD₂₈₀.
3. Calculate the RNA concentration using the following equation:

Appendix

Concentration measurement
RNA (and DNA) has an absorbance peak at 260nm. The ratio A₂₆₀:A₂₈₀ is an indication of the purity of the RNA. The ratio (for pure RNA) should be 1.8 - 2.1. However, even if the ratio is less than 1.8, the RNA quality may be acceptable and can be confirmed by the subsequent gel electrophoresis.
Alternatives
Trizol can be purchased from GibcoBRL. If you wish to avoid phenol, many companies sell RNA extraction kits (e.g. Qiagen, Hybaid, etc).

Handling RNA -the hazards of RNase
One of the major difficulties in working with RNA is its susceptibility to hydrolytic cleavage by the enzyme ribonuclease (RNase). RNase is literally everywhere - it is secreted from the skin and tends to coat tubes, benches, pipettes an so on. It is also very stable Ð it retains its activity even after autoclave procedures and cannot be removed by cleaning surfaces with ethanol or isopropanol. It even retains activity after exposure to chaotropic agents such as guanidium thiocyanate. The only way to eradicate the effect of RNase is to chemically inactivate it. This requires the use of the methylating agent, diethylpyrocarbonate (DEPC), which irreversibly modifies the active site histidine residue of the enzyme, thereby inactivating it. All water used for RNA work must be treated with DEPC before use. DEPC is dispensed in a fume hood and added to water in an autoclavable (Schott type) bottle containing a stir bar. The water is stirred for one hour in the fume cupboard and after treatment the solution is autoclaved to denature the DEPC. To avoid contamination by RNase, it is important to handle solutions and material for RNA use with extra care:
-Wear gloves at all times
-Maintain separate stocks of chemicals reagents for RNA work - do not use for other laboratory stocks. Many suppliers can provide RNase-free chemicals and reagents.
-Solutions that have been prepared for RNA work should be kept separate from the rest of the laboratory solutions- label these reagents appropriately.
-All pipette tips and Eppendorf tubes should not be handled by hand prior to autoclaving. If possible, purchase pre-packed RNase free materials.

First-strand cDNA Synthesis

Prepare in a sterile tube:

template RNA:

total RNA  0.1-5µg

or poly(A)+ mRNA  10ng-0.5µg,

or specific RNA  0.01pg-0.5µg

primer:

oligo(dT)18   0.5µg

or random hexamer 0.2µg,

or sequence-specific 15-20pmol,

deionized water (nuclease free) up to 11µl.

Incubate the mix at 70℃for 5 minutes and chill on ice.

Add the following in the order indicated:

5X reaction buffer 4µl,

10mM 4 dNTP mix 2µl (1.0mM - final concentration),

ribonuclease inhibitor 20u,

deionized water (nuclease free) to 19µl.

Incubate at 37℃for 5 minutes. If random primer is used, incubate at 25℃for 5 minutes.

Add 40 units of M-MuLV Reverse Transcriptase . Incubate the reaction mixture, containing oligo(dT)18 or sequence-specific primer at 37℃for 60 minutes. If using random hexamer primer, incubate at 25℃for 10 minutes and then at 37℃for 60 minutes.

Stop the reaction by heating at 70℃for 10 minutes. Chill on ice.

Note

The synthesized cDNA can be amplified by the PCR (see Protocols for PCR using Taq and Pfu DNA Polymerases) without intermediate phenol/chloroform extraction or ethanol precipitation.

Reference

Gerard, G.F. and D'Alessio, I.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.