CytoSelect™ 96-Well Hematopoietic Colony Forming Cell Assay

Catalog No. ABIN2344936

Product Details

Reactivity: Mammalian

Application: Biochemical Assay (BCA)

Brand: CytoSelect™

Sample Type: Cell Samples

Characteristics: CytoSelect™ 96-well Hematopoietic Colony Forming Cell Assay does not involve subjective manual counting of colonies or require a 2-3 week incubation period. Instead cells are incubated only 7-10 days in a semisolid methycellulose media before being solubilized, lysed and detected by the patented CyQuant® GR Dye in a fluorescence plate reader (see Assay Principle below). Alternatively, viable CFCs can be easily recovered for further culturing and testing. This format provides a quantitative, high-throughput method to accurately measure HSC or hematopoietic progenitor clonogenic capability.

Components:

1. 4X Lysis Buffer : One 10 mL bottle
2. CyQuant® GR Dye : One 75 μL tube
3. CytoSelect™ Methylcellulose Medium : One sterile 15 mL bottle of 1% Methyl- cellulose, 25% FBS, 2% BSA, 0.15% NaHCO3, 50 μM 2-mercaptoethanol in Iscove's MDM

Material not included:

1. Cells and Culture Medium
2. Cell Resuspension Media (IMDM containing 25 % FBS)
3. Cytokine/Growth Factor Supplements
4. 1X PBS
5. 37 °C Incubator, 5 % CO2 Atmosphere
6. Light Microscope
7. 96-well Fluorometer
8. 96-well Tissue Culture Plate 4

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Methylcellulose medium supports growth of hematopoietic cell colonies
• Fully quantify hematopoietic stem cells with no manual cell counting
• Results in 7-10 days, not 3 weeks

Assay Time: 7 - 10 d

Reagent Preparation:

• Thaw CytoSelect™ Methylcellulose Medium at 4 °C overnight. Mix the media well prior to aliquoting or use, ensuring a homogenous solution. Due to its viscosity, let the media to sit for 15 minutes to allow bubbles to rise to the top.

Assay Procedure:

I. Hematopoietic Colony Forming Cell Assay (under sterile conditions)

1. Harvest cells in cell resuspension medium (see Materials Not Supplied) at 1 - 5 x 105 cells/mL. Desired cytokines and growth factors should be added directly to the cell suspension. Note: Cytokine/growth factors will be diluted 10-fold by addition of the CytoSelect™ Methylcellulose Medium.
2. Immediately dispense 15 μL of Cell Suspension containing growth factors into each well of a 96-well tissue culture plate.
3. Add 135 μL of CytoSelect™ Methylcellulose Medium (see Preparation of Reagents) to each well. Pipette each well 7-10 times to mix thoroughly. Notes: • Try to avoid adding air bubbles to the well. • To avoid fast and uneven evaporation that leads to aberrant results, we suggest not using the wells on the plate edge, or filling the edge wells with medium to reduce evaporation.
4. Incubate the cells for 7-10 days at 37 °C and 5 % CO2. Examine the colony formation under a light microscope.

II. Quantitation of Colony Formation (skip to section III if cell recovery/re-plating is desired)

1. Prepare sufficient 4X Lysis Buffer/CyQuant® GR dye solution for all samples by diluting the dye 1:75 in 4X Lysis Buffer (for example, add 5 μL dye to 370 μL of 4X Lysis Buffer).
2. Add 50 μL of 4X Lysis Buffer/CyQuant® GR dye solution to each well (already containing 150 μL of solution). Pipette each well 7-10 times to ensure a homogeneous mixture. Incubate the plate at room temperature for 30 minutes.
3. Transfer 100 μL of the mixture to a 96-well plate suitable for fluorescence measurement.
4. Read the plate in a 96-well fluorometer using a 485/520 nm filter set.

III. Cell Recovery and Re-plating (under sterile conditions)

1. Add 150 μL of cold, sterile culture medium to each well.
2. Pipette each well 10-12 times to mix thoroughly.
3. Transfer the entire mixture to at least 20 volumes of standard culture medium (for example, 1 mL would be transferred to 20 mL media).
4. Centrifuge the cell pellet and aspirate the media supernatant.
5. Resuspend the pellet and transfer to a tissue culture flask or dish. 5
6. Transfer to a cell culture incubator. Cell Dose Curve (Optional)
   1. Harvest cells in cell resuspension medium at 1 - 5 x 106 cells/mL.
   2. Prepare a serial 2-fold dilution in resuspension medium, including a blank without cells.
   3. Transfer 150 μL of each dilution to a 96-well plate.
   4. Add 50 μL of 4X Lysis Buffer/CyQuant® GR dye solution to each well (already containing 150 μL of solution). Pipette each well 7-10 times to ensure a homogeneous mixture. Incubate the plate at room temperature for 30 minutes.
   5. Transfer 100 μL of the mixture to a 96-well plate suitable for fluorescence measurement.
   6. Read the plate in a 96-well fluorometer using a 485/520 nm filter set.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: 4 °C/-20 °C

Storage Comment: Store the CytoSelect™ Methylcellulose Medium at -20°C. If the methylcellulose medium will not be used all at once, it should be aliquoted to avoid multiple freeze/thaw cycles. Store all other components at 4°C. 4

References

Neri, Ren, Gratton, Stebner, Johnson, Klimowicz, Duggan, Tassone, Mansoor, Stewart, Lonial, Boise, Bahlis: "Bortezomib-induced "BRCAness" sensitizes multiple myeloma cells to PARP inhibitors." in: Blood, Vol. 118, Issue 24, pp. 6368-79, (2011) (PubMed).

Target Details

Background: Hematopoietic stem cells (HSC) are well-characterized, tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long maintenance of the hematopoietic system. HSC are rare cells that reside in adult bone marrow where hematopoiesis is continuously taking place, but can also be found in cord blood, fetal liver, adult spleen and peripheral blood. Throughout the life span, hematopoiesis continuously replenishes the various lymphoid, myeloid and erythroid-megakaryocyte lineages, but also to maintain a small pool of HSC with the self- renewal capacity that is capable of carrying on hematopoiesis. From HSC to mature blood cells, extensive proliferation and expansion occurs that results in the production of millions of blood cells. When cultured in a suitable semi-solid matrix, such as methylcellulose supplemented with nutrients and cytokines, HSC or hematopoietic progenitors called colony-forming cells (CFCs) proliferate to form discrete cell clusters or colonies. Colony types include colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), CFU-granulocyte, macrophage (CFU-GM) and CFU- granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM). In classical CFC assays, culture usually takes place in a 35 mm dish for 14-21 days for the colonies to reach certain size (> 40 cells/colony) for manual counting. The CFCs are classified and manually enumerated based on the morphological recognition of one or more types of hematopoietic lineage cells within the colony.