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

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

Western Blotting Tips

1) Load 20 to 35 μl of your samples unless you know for a fact that this is too much.

2) In the first lane load 25 μl of positive bloting control. This is lysate stimulated with PMA or LPA. This is good for most CCL39 blots.

3) Molecular Wt Markers - There are now two kinds. One is the BioRad Prestained standards we've always used. The other is MagicMarker. This is a non-stained protein that has an IgG recognition site. That means each of the proteins will bind secondary antibody and will be visible on the film. To use this:

- In the second lane load 10-15 μl of the BioRad standard

- In the SAME well/lane add 3 μl of the MagicMarker standard (found in a box in the

freezer in the main room

4) Run the gel to the bottom but not off. Make certain the dye is just coming to the edge of the glass not the gasket. Remember, 200 v for 1 hour is an approximation - you may very likely have to turn it up a little more. If this is a key experiment, run the gel at 100 v for 10-15 min first then run for 200V. The proteins will be tighter. ALSO if the % cross-linking is low then the proteins will run faster than the higher % gels

5) Soak the gel in transfer buffer for a minute or two to reduce salt that can reduce transfer.

6) Cut the paper to fit the gel, unless you are using ERK or p-ERK. Remember unless we have done the blot many times you need to show the molecular weights to prove that the band you think is your protein is the right one. Clip the corner of the blot as directed in the protocol. Not all antibodies are as specific as some of the ones we use. Always mark the film with the markers!

7) The fit of the gel sandwich should be tight and free of air bubbles. If the pads are getting

thin add a third to make certain there is proper contact between paper and gel.

8) Transfer at 90 V for 1 hr unless using a 12-16% gel then add 15 min

9) Some antibodies need a different washing solution. The concentration or type of detergent (Tween-20) may not work for some. If using a new antibody, check!

10) Block in the BioRad dry milk, not the carnation or other generic instant milk. Use the same concentration as before. We get high backgrounds with some of these other milks. Some antibodies need BSA instead of milk to block with. Always look up new antibody requirements.

11) When starting use 1:500 dilutions for the primary and 1:1000 for the secondary. Use only the antibodies from the "Chinese take-out" cool-safe box

12) Don't let the blot get too dry - this may have been a problem

13) Do a 10 sec, 1 min and 10 min exposure.

Purifying DNA from TAE agarose gels

1. Excise band from agarose gel.

2. Add 3 volumes of NaI and incubate at 55°C to melt gel.

3. Add 5μl GLASSMILK suspension.

4. Pellet GLASSMILK/DNA complex (5 seconds).

5. Wash pellet with 1ml NEW Wash.

6. Pellet again and rewash with 0.5ml NEW Wash.

7. Elute DNA with 10-20μl water or 0.1x TE.

Proteolytic enzymes

Trypsin, collagenase, or pronase, usually in combination with EDTA, causes cells to detach from the growth surface. This method is fast and reliable but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum.

Lipid siRNA transfection

For some cell lines, either electroporation or cationic lipids may be used. Basically any tfx protocol will require you to work out conditions for every cell line. Sometimes between 70-90% or more cells can be transfected with cationic lipids. With a bit of tweaking, and depending on the cell line, you can drive siRNAs into almost 100% of the cells with the following protocol:

Seed cells high, so that they are 85%+ on the day of transfection:

For one 10 cm dish:

40 ul Lipofectamine 2000

20 ug DNA

100-300 pmoles siRNA

tube 1

add the siRNA and/or DNA to 2 ml SF-DMEM (no drugs added)

tube 2

add the lipid to 2 ml SF-DMEM (no drugs added)

mix these two tubes immediately, for a volume of 4 ml, and then add to cells which have been washed 2X with SF-DMEM. Incubate 3-6 hours, then add normal media. Check for expression or knockdown 36+ hours post-transfection. Knockdown by siRNAs usually last 3-5 cell doublings.

When oligofectamine is used, the identical protocol is used, but:

Seed cells at 15% confluency, perform tfx with 30 ul oligofectamine and siRNAs, then repeat tfx 36 hours later. Analysis is performed when cells are 85-90% confluent. I have not calculated the transfection efficiency with oligofectamine.

ELISPOT Protocol

Coat the Plate:
1. Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s
LEAF™ format antibodies are specifically designed for this assay) to a
final concentration of 0.5–4 μg/ml in sterile Coating Buffer and transfer 100
μl/well to a high affinity binding PVDF membrane ELISPOT plate (e.g., Millipore;
Cat. No. MAIPS-4510).
2. Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours in
humidified atmosphere.

Block the Plate:
3. Wash plate 3 times with sterile PBS, 200 μl/well.
4. Add 200 μl/well of sterile Blocking Buffer.
5. Seal plate and incubate at room temperature for ≥ 1 hour.
6. Wash plate 3 times with sterile PBS, 200 μl/well.
Set-Up Tissue Culture and Add Antigen or Mitogen:
7. Add appropriate sterile antigen or mitogen solution diluted in appropriate
sterile tissue culture medium (TC) to ELISPOT plate, 100 μl/well.
8. Add cells diluted in sterile TC medium, 100 μl/well. Use 50,000-500,000 cells/
well (the minimum number of cells should be determined in preliminary
experiments).
9. Seal plate and incubate at 37°C 5% CO2 in humidified atmosphere for the
optimum stimulation period. BioLegend recommends a 24 hour incubation
for IFN-γ, IL-2, and TNF-α, and a 48 hour incubation period for IL-4, IL-5, and
IL-10 for most activation conditions.

Add Detection Antibody:
10. Wash plate 3 times with PBS, 200 μl/well.
11. Wash plate 3 times with PBS-Tween, 200 μl/well.
12. Add 100 μl/well of diluted biotinylated detection antibody at 0.25-2 μg/ml in
PBS-Tween-BSA.
13. Seal the plate and incubate at 4°C overnight, or 2 hr at room temperature.

Add Avidin-Horseradish Peroxidase (Av-HRP):
14. Wash plate 4 times with PBS-Tween, 200 μl/well.
15. Add 100 μl per well of the Av-HRP conjugate (Cat. No. 405103) or other
enzyme conjugate diluted to its pre-determined optimal concentration in
PBS-Tween-BSA (usually between 1/500 – 1/2000).

Source: 1. postech.ac.kr

2.http://www.biolegend.com/media_assets/support_protocol/BioLegend_ELISPOT_protocol.pdf