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Anti CD44 v9 and CD44 v10-e16 Antibody Protocol

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Flow cytometry Protocol (Cell Analysis)

A. Cell Preparation

1. Remove cells from incubator.

2. Discard culture medium.

3. Briefly rinse the cell layer with PBS.

4. Add 0.25% trypsin-EDTA solution to dish. Return the dish to the incubator and incubate for 2-10 minutes or until cells are detached.

5. Resuspend cells in complete growth medium to inactivate the trypsin.

B. Staining

1. Aliquot 1x105 cells into each assay tube.

2. Add 150 μl 0.2 % BSA in PBS to each tube and rinse by centrifugation.

3. Add 50 μl diluted primary antibody (3 μg/ml RV3 in 0.2 % BSA in PBS) to the assay tubes.

4. Incubate 45 minutes at 4°C.

5. Add 100 μl 0.1 % BSA in PBS to each tube and wash by centrifugation.

6. Wash two times in 150 μl 0.1 % BSA in PBS by centrifugation.

7. Resuspend cells in 50 μl PE-labeled secondary antibody solution (Jackson Immuno Research 712-116-153),diluted 1:200 in 0.1% BSA in PBS.

8. Incubate 30 minutes at 4 °C in the dark.

9. Add 100 μl 0.1 % BSA in PBS to each tube and wash by centrifugation.

10. Wash two times in 150 μl 0.1 % BSA in PBS by centrifugation.

11. Resuspend cells in 100 μl PBS.

12. Add 100 μl Propidium Iodide (SIGMA, P4864), diluted 1:500 in PBS, to stain dead cells.

13. Analyze using flow cytometry.

Flow cytometry Protocol (Cell Sorting)

A. Cell Preparation

1. Prepare cultured cells for sortingbased on the ratio of the CD44v expression cells.

2. Remove cells from incubator.

3. Discard culture medium.

4. Briefly rinse the cell layer with PBS.

5. Add 0.25% trypsin-EDTA solution to dish. Return the dish to the incubator and incubate for 2-10 minutes or until cells are detached.

6. Resuspend the cells in complete growth medium to inactivate the trypsin.

B. Staining (for 1x107 cells)

1. Aliquot 1x107 cells into 15ml tube.

2. Add 10 ml 0.2 % BSA in PBS to the tube andrinseby centrifugation.

3. Add 5ml diluted primary antibody (3 μg/ml RV3 in 0.2 % BSA in PBS) to the tube.

4. Incubate with gentle agitation 45 minutes at 4 °C.

5. Wash three times in 10 ml 0.1 % BSA in PBS by centrifugation.

6. Resuspend cells in 5ml PE-labeled secondary antibody solution (Jackson Immuno Research 712-116-153), diluted 1:200 in 0.1 % BSA in PBS.

7. Incubate 45 minutes at 4 °C in the dark.

8. Wash three times in 10 ml 0.1 % BSA in PBS by centrifugation.

9. Resuspend cellsin5ml PBS.

10. Add 5ml Propidium Iodide (SIGMA, P4864) diluted 1:500 in PBS, to stain dead cells.

11. Sort CD44v high and low expression cells using a cell sorter.

12. Wash the sorted cells in 5 ml complete growth medium (added antibiotic drug)threetimes by centrifugation.

13. Culture the sorted cells and scale up.
・ Note the passage number and analyze the cell population periodically using flow cytometry.

14. If desired, sort the cells again, they would be high-enrichment.

Immunohistochemistry Protocol (Paraffin)

A. Deparaffinization / Rehydration

1. Deparaffinize/hydrate
  a. Incubate sections in xylene three times for 5 minutes each.
  b. Incubate sections in 100% ethanol for 10 seconds.
  c. Incubate sections in 90% ethanol for 10 seconds.
  d. Incubate sections in 70% ethanol for 10 seconds.

2. Wash sections in dH2O for 5 minutes.

B. Antigen Unmasking

1. Immerse sections in 10 mM citrate buffer (pH 6.0) for 7 minutes at 100 °C in microwave.

2. Coolslides on bench top for 30 minutes.

3. Wash sections in TBS-T buffer for 5 minutes.

C. Staining

1. Incubate sections in 0.3% hydrogen peroxide in Methanol for 15 minutes to block endogenous peroxidase.

2. Wash sections in TBS-T buffer threetimes for 5 minutes each.

3. Cover sections with 300 μl blocking solution (10 % normal rabbit serum in TBS) for 30 minutes at room temperature.

4. Remove excess blocking solution and add 200 μl diluted primary antibody to each section (0.2μg/mlRV3 in 1.5 % normal rabbit serum in TBS). Incubate 1 hour at room temperature.

5. Remove antibody solution and wash sections in TBS-T buffer three times for 5 minutes each.

6. Add 200 μl biotinylated secondary antibody (Dako, E0468), diluted 1:200in 1.5 % normal rabbit serum in TBS, to each section.

7. Incubate 30 minutes at room temperature.

8. Remove antibody solution and wash sections in TBS-T buffer three times for 5 minutes each.

9. Add 200 μl VECTASTAIN ABC Reagent (VECTOR LABORATORIES,PK-6100) to each section. Incubate 30 minutes at room temperature.

10. Wash sections in TBS-T buffer three times for 5 minutes each.

11. Add 200 μl peroxidase substrate reagent (PIERCE, 34065) to each section and incubate until desired stainintensity develops (about 1 minute).

12. Wash sections in TBS-T buffer three times for 5 minutes each.

13. Wash sections in dH2O two times for 5 minutes each.

14. Counterstain sections in hematoxylin per manufacturer’s instructions.

15. Dehydratesections:
  a. Incubate sections in 70% ethanol for 10 seconds.
  b. Incubate sections in 90% ethanol for 10 seconds.
  c. Incubate sections in 100% ethanol for 10 seconds.
  d. Incubate sections in xylene three washes for 5 minutes each.

16. Mount coverslips.

Immunofluorescence Protocol

A. Cell Preparation

1. Grow cultured cells on Cell Chamber Slide

2. Remove cells from incubator.

3. Discard medium and rinse briefly with PBS.

4. Fix in 4% paraformaldehyde in PBS for 15 minutes at room temperature.

5. Remove fixative and rinse three times in PBS for 3 minutes each.

B. Staining

1. Block sample in 3% BSA in PBS for 30 minutes at room temperature.

2. Remove excess blocking solution and apply diluted primaryantibody (3μg/ml RV3 in 0.2 % BSA in PBS).

3. Incubate 1 hour at room temperature.

4. Wash three times in PBS for 3 minutes each.

5. Apply Alexa Fluor 488 anti-rat IgG secondary antibody solution (Invitrogen, A11006), diluted 1:400 in 0.2 % BSAin PBS.

6. Incubate 1 hour at room temperature in the dark.

7. Wash three times in PBS for 3 minutes each.

8. Mount the coverslip using an anti-fade mounting reagent with DAPI (Invitrogen, S36939).

9. Seal slide by painting around edges of coverslip with nail polish.

10. Store slides flat at 4 °C protected from light until until examined.

Western blot Protocol

1. Load 20 μl cell lysate samples (40μg/lane) onto SDS-PAGE gel (ATTO E-T10L).

2. Electrotransfer to PVDF membrane.

3. Block membrane in 5% Skim milk in PBS-T for 1 hour at room temperature.

4. Incubate membrane with 1 μg/ml RV3 primary antibody in Can Get Signal Solution I (TOYOBO, NKB201) with gentle agitation overnight at 4°C.

5. Wash membrane in PBS-T three times for 5 minutes each.

6. Incubate membrane with HRP-conjugated anti-rat IgG secondary antibody (GE Healthcare NA9350V), diluted 1:20000 in Can Get Signal Solution II (TOYOBO, NKB301), with gentle agitation 30 minutes at 37 °C.

7. Washmembrane in PBS-T three times for 5 minutes each.

8. Incubate membrane in detection mix (Thermo SuperSignal West Dura, 34075) with gentle agitation 1 minute at room temperature.

9. Detect using an imager.

Product List

Product Name Cat# Quantity Price

Anti CD44v9

CAC-LKG-M003 50UG

¥ 60,000
$ 800
€ 600

Anti CD44v9

CAC-LKG-M001 100UG

¥ 100,000
$ 1334
€ 1000

Anti CD44v10-e16

CAC-LKG-M002 100UG

¥ 100,000
$ 1334
€ 1000

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.