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Human Methylcellulose Complete Media

Catalog #: HSC003
29 Citations Datasheet
Catalog # Availability Size / Price Qty
HSC003
Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. 
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Citations (29)
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Human Methylcellulose Complete Media Summary

Kit Summary

For the differentiation and enumeration of human hematopoietic stem cells, optimized with premium quality cytokines.

Key Benefits

  • Does not require addition of serum or cytokines
  • Optimized with premium quality recombinant proteins
  • Excellent optical clarity facilitates colony identification
  • High lot-to-lot consistency decreases variation
 

 

Why use R&D Systems Human Methylcellulose Complete Media for Colony Forming Cell Assays?

Colony forming cell (CFC) assays, which are used to enumerate and quantify multi-potent and single lineage hematopoietic progenitors, can be time consuming and laborious.

Successful growth and enumeration of cell colonies is dependent on factors such as accurate cell counts, the presence of growth factors and/or cytokines, adequate humidity, and the use of high quality media. R&D Systems offers Human Methylcellulose Complete Media, which contains all growth factors and cytokines needed to support optimal colony growth and enumeration. The Human Methylcellulose Complete Media is specially formulated and has been optimized for CFC assays using burst-forming and colony-forming erythroid (BFU-E, CFU-E), myeloid (CFU-GM, CFU-G, CFU-M), and mixed lineage (CFU-GEMM) progenitors of human origin. This product can also be used in the long-term culture-initiating cell (LTC-IC) assay.

R&D Systems Human Methylcellulose Complete Media:

  • Optical clarity facilitates colony identification.
  • High lot-to-lot consistency decreases variation.
  • Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
  • Increased cloning efficiency and improved colony growth compared to agar.
  • Does not require addition of serum or cytokines.
 

 

Kit Contents

  • 100 mL of Human Methylcellulose Complete Media and 15 mL of Cell Resuspension Solution.

Contents Concentration
(when diluted to a final volume of 100 mL)
Methylcellulose (1500 cps) in
Iscove’s Modified Dulbecco's Medium		 1.4%
Fetal Bovine Serum 25%
Bovine Serum Albumin 2%
L-Glutamine 2 mM
2-Mercaptoethanol 5 x 10-5 M
Recombinant Human SCF 50 ng/mL
Recombinant Human GM-CSF 10 ng/mL
Recombinant Human IL-3 10 ng/mL
Recombinant Human Epo 3 IU/mL

 

Cell Resuspension Solution (15 mL)

Contents Concentration
Fetal Bovine Serum in
Iscove’s Modified Dulbecco’s Medium		 50%

Stability and Storage

Human Methylcellulose Complete Media and the Cell Resuspension Solution should be stored at ≤-20 °C upon receipt. Storage at 2 °C to 8 °C is not recommended.

Precautions

The acute and chronic effects of overexposure to this media are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling this media.

Limitations

  • The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
  • The reagent should not be used beyond the expiration date indicated on the label.
  • Human hematopoietic progenitors derived from different individuals may cause results to vary.

 

 

 

Guide to Choosing Media for the Colony Forming Cell (CFC) Assay

Human Methylcellulose Stock and Base Media

Catalog # Product Description Volume Colonies Selected for Contains Serum Cytokines Included
HSC001 Methylcellulose Stock Solution 100 mL N/A* No None
HSC002 Human Methylcellulose
Base Media		 90 mL N/A* Yes None
HSC011 StemXVivo® Methylcellulose
Concentrate		 50 mL N/A* No None

Complete Human Methylcellulose Media

Catalog # Product Description Volume Colonies Selected for Contains Serum Cytokines Included
HSC003 Human Methylcellulose Complete Media 100 mL BFU-E
CFU-E
CFU-G
CFU-GEMM
CFU-GM
CFU-M		 Yes Epo
GM-CSF
IL-3
SCF
HSC004 Human Methylcellulose Complete Media without Epo 100 mL CFU-G
CFU-GM
CFU-M		 Yes SCF
GM-CSF
IL-3
HSC005 Human Methylcellulose
Enriched Media		 100 mL BFU-E
CFU-E
CFU-G
CFU-GEMM
CFU-GM
CFU-M		 Yes Epo
G-CSF
GM-CSF
IL-3
IL-6
SCF
HSC005SF Human Methylcellulose
Serum-Free Enriched Media		 100 mL BFU-E
CFU-E
CFU-G
CFU-GEMM
CFU-GM
CFU-M		 No Epo
G-CSF
GM-CSF
IL-3
IL-6
SCF

*Base media and stock solutions do not contain cytokines and will not support colony growth unless conditioned media, cytokines, or other culture supplements are added.

Specifications

Source
N/A
Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Human

Product Datasheets

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Product Datasheet

Scientific Data

Cell Morphology Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay.  View Larger

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay.  Colony forming unit-erythroid (CFU-E) are clonogenic progenitors that produce only one or two clusters with each cluster containing from 8 to approximately 100 hemoglobinized erythroblasts. It represents the more mature erythroid progenitors that have less proliferative capacity.B. Colony forming unit-granulocyte (CFU-G) are clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils, or neutrophils.C. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. The morphology is similar to the CFU-M and CFU-G descriptions.D. Burst forming unit-erythroid (BFU-E) colonies can be described as small (3 to 8 clusters), intermediate (9 to 16 clusters), or large (more than 16 clusters) according to the number of clusters present. These are primitive erythroid progenitors that have high proliferative capacity.E. Colony forming unit-macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages.F. Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) are multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages, as the name indicates.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Human Methylcellulose Complete Media is used in the Colony Forming Cell Assay using the following procedure:

  • Prepare human mononuclear cells
  • Add cells to Human Methylcellulose Complete Media
  • Plate and incubate cells
  • Identify and count colonies

View Human Procedure
 

 

Reagents Provided

Reagents supplied in the Human Methylcellulose Complete Media (Catalog # HSC003):

  • 100 mL of Human Methylcellulose Complete Media and 15 mL of Cell Resuspension Solution.

Contents Concentration
(when diluted to a final volume of 100 mL)
Methylcellulose (1500 cps) in
Iscove’s Modified Dulbecco's Medium		 1.4%
Fetal Bovine Serum 25%
Bovine Serum Albumin 2%
L-Glutamine 2 mM
2-Mercaptoethanol 5 x 10-5 M
Recombinant Human SCF 50 ng/mL
Recombinant Human GM-CSF 10 ng/mL
Recombinant Human IL-3 10 ng/mL
Recombinant Human Epo 3 IU/mL

Cell Resuspension Solution (15 mL)

Contents Concentration
Fetal Bovine Serum in
Iscove’s Modified Dulbecco’s Medium		 50%

 

Other Supplies Required

Reagents

  • Cells derived from bone marrow, blood, or enriched CD34+ cells
  • Iscove's Modified Dulbecco's Media (IMDM)
  • Ca2+/Mg2+-free Hank's Balanced Salt Solution (HBSS)
  • Ficoll-Paque™ PLUS (GE Healthcare) or equivalent

Materials

  • 100 mm culture plates
  • 35 mm culture plates
  • 15 mL centrifuge tubes
  • 50 mL centrifuge tubes
  • 10 mL syringes
  • 3 mL syringes
  • 5 mL vials
  • 16 gauge 1½ inch needle
  • 14 gauge laboratory pipetting needle
  • Heparinized syringes or Vacutainers®
  • Serological pipettes
  • Pipettes and pipette tips

Equipment

  • 37 °C and CO2 humidified incubator
  • Centrifuge
  • Vortex mixer
  • Hemocytometer
  • Inverted Microscope

 

Procedure Overview

Prepare mononuclear cells by Ficoll-Paque gradient centrifugation.

Wash the cells two times with HBSS and pool the cells.

Centrifuge the cells at 400 x g for 10 minutes.

Prepare mononuclear cells by Ficoll-Paque gradient centrifugation

Thaw aliquots of Human Methylcellose Complete Media at room temperature.

Thaw aliquots of Methylcellulose Stock Solution at room temperature

Resuspend mononuclear cells in 10 mL of IMDM.

Thaw aliquots of Methylcellulose Stock Solution at room temperature

Perform a cell count.

Perform a cell count

Transfer the appropriate volume of cells plus a slight excess into a new 15 mL centrifuge tube.

Centrifuge at 300 x g for 10 minutes.

Transfer the appropriate volume of cells plus a slight excess into a new 15 mL centrifuge tube

Remove the supernatant.

Resuspend the cells in Cell Resuspension Solution to the desired stock cell number to generate a 10X stock concentration.

Remove the supernatant

Combine the appropriate volume of 10X cell stock with the desired cell culture supplements/cytokines, and Human Methylcellulose Base Media. The final methylcellulose concentration should be 1.27%.

Combine the appropriate volume of 10X cell stock

Vortex the samples vigorously.

Wait approximately 20 minutes to allow air bubbles to escape.

Add 1.1 mL of the cell mixture to a 35 mm culture plate using a 3 mL syringe and a 16 gauge needle.

Spread the media evenly by gently rotating the plate.

Vortex the samples vigorously

Place two 35 mm plates into a 10 cm plate.

Add one uncovered 35 mm plate that contains 3-4 mL of sterile water.

Cover the 10 cm plate and place it in a 37 °C and 5% CO2 incubator.

Incubate the cells for 14-16 days.

Place two 35 mm plates into a 10 cm plate

Use an inverted microscope and a scoring grid to identify and count individual colonies.

Place two 35 mm plates into a 10 cm plate

Citations for Human Methylcellulose Complete Media

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

29 Citations: Showing 1 - 10
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  1. A novel LGALS1-depended and immune-associated fatty acid metabolism risk model in acute myeloid leukemia stem cells
    Authors: Qin, H;Peng, M;Cheng, J;Wang, Z;Cui, Y;Huang, Y;Gui, Y;Sun, Y;Xiang, W;Huang, X;Huang, T;Wang, L;Chen, J;Hou, Y;
    Cell death & disease  2024-07-05
  2. 8-Cl-Ado and 8-NH2-Ado synergize with venetoclax to target the methionine-MAT2A-SAM axis in acute myeloid leukemia
    Authors: Guo, J;Buettner, R;Du, L;Li, Z;Liu, W;Su, R;Chen, Z;Che, Y;Zhang, Y;Ma, R;Nguyen, LXT;Moore, RE;Khyatiben, P;Chen, MH;Patrick, P;Wu, X;Marcucci, G;Wang, L;Horne, D;Chen, J;Yang, Y;Rosen, ST;
    Leukemia  2024-04-20
  3. Effect of the LSD1 inhibitor RN-1 on ?-globin and global gene expression during erythroid differentiation in baboons (Papio anubis)
    Authors: Ibanez, V;Vaitkus, K;Ruiz, MA;Lei, Z;Maienschein-Cline, M;Arbieva, Z;Lavelle, D;
    PloS one  2023-12-22
  4. ZAK?/P38 kinase signaling pathway regulates hematopoiesis by activating the NLRP1 inflammasome
    Authors: Rodríguez-Ruiz, L;Lozano-Gil, JM;Naranjo-Sánchez, E;Martínez-Balsalobre, E;Martínez-López, A;Lachaud, C;Blanquer, M;Phung, TK;García-Moreno, D;Cayuela, ML;Tyrkalska, SD;Pérez-Oliva, AB;Mulero, V;
    EMBO molecular medicine  2023-09-07
  5. The Role of Cytokine-Inducible SH2 Domain-Containing Protein (CISH) in the Regulation of Basal and Cytokine-Mediated Myelopoiesis
    Authors: Naser, W;Maymand, S;Dlugolenski, D;Basheer, F;Ward, AC;
    International journal of molecular sciences  2023-08-14
  6. Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring
    Authors: MJ Nash, E Dobrinskik, TK Soderborg, RC Janssen, DL Takahashi, TA Dean, O Varlamov, JD Hennebold, M Gannon, KM Aagaard, CE McCurdy, P Kievit, BC Bergman, KL Jones, EM Pietras, SR Wesolowski, JE Friedman
    Cell Reports, 2023-04-13;42(4):112393.  2023-04-13
  7. Tumorigenic role of Musashi-2 in aggressive mantle cell lymphoma
    Authors: M Sureda-Góm, P Balsas, ML Rodríguez, F Nadeu, A De Bolòs, Á Eguileor, M Kulis, G Castellano, C López, E Giné, S Demajo, P Jares, JI Martín-Sub, S Beà, E Campo, V Amador
    Leukemia, 2022-12-12;0(0):.  2022-12-12
  8. Chromatin-associated orphan snoRNA regulates DNA damage-mediated differentiation via a non-canonical complex
    Authors: C Han, LY Sun, XQ Luo, Q Pan, YM Sun, ZC Zeng, TQ Chen, W Huang, K Fang, WT Wang, YQ Chen
    Cell Reports, 2022-03-29;38(13):110421.  2022-03-29
  9. YTHDF2 is a potential target of AML1/ETO-HIF1&alpha loop-mediated cell proliferation in t(8;21) AML
    Authors: Z Chen, YL Shao, LL Wang, J Lin, JB Zhang, Y Ding, BB Gao, DH Liu, XN Gao
    Oncogene, 2021-05-06;0(0):.  2021-05-06
  10. Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells
    Authors: CL Jones, BM Stevens, DA Pollyea, R Culp-Hill, JA Reisz, T Nemkov, S Gehrke, F Gamboni, A Krug, A Winters, S Pei, A Gustafson, H Ye, A Inguva, M Amaya, M Minhajuddi, D Abbott, MW Becker, J DeGregori, CA Smith, A D'Alessand, CT Jordan
    Cell Stem Cell, 2020-08-20;0(0):.  2020-08-20
  11. Acute myeloid leukemia cells secrete microRNA-4532-containing exosomes to mediate normal hematopoiesis in hematopoietic stem cells by activating the LDOC1-dependent STAT3 signaling pathway
    Authors: C Zhao, F Du, Y Zhao, S Wang, L Qi
    Stem Cell Res Ther, 2019-12-16;10(1):384.  2019-12-16
  12. PM2.5 collecting in a tire manufacturing plant affects epithelial differentiation of human umbilical cord derived mesenchymal stem cells by Wnt/beta-catenin pathway
    Authors: J Yan, L Jin, D Lin, CH Lai, Z Xu, R Wang, YC Chen, B Hu, CH Lin
    Chemosphere, 2019-11-22;244(0):125441.  2019-11-22
  13. Ceramide analog SACLAC modulates sphingolipid levels and Mcl-1 splicing to induce apoptosis in acute myeloid leukemia
    Authors: JM Pearson, SF Tan, A Sharma, C Annageldiy, TE Fox, JL Abad, G Fabrias, D Desai, S Amin, HG Wang, MC Cabot, DF Claxton, M Kester, DJ Feith, TP Loughran
    Mol. Cancer Res., 2019-11-19;0(0):.  2019-11-19
  14. The novel Isatin analog KS99 targets stemness markers in acute myeloid leukemia
    Authors: C Annageldiy, K Gowda, T Patel, P Bhattachar, SF Tan, S Iyer, D Desai, S Dovat, DJ Feith, TP Loughran, S Amin, D Claxton, A Sharma
    Haematologica, 2019-05-23;0(0):.  2019-05-23
  15. Effects of continuous high-dose G-CSF administration on hematopoietic stem cell mobilization and telomere length in patients with amyotrophic lateral sclerosis - a pilot study
    Authors: S Iberl, AL Meyer, G Müller, S Peters, S Johannesen, I Kobor, F Beier, TH Brümmendor, C Hart, R Schelker, W Herr, U Bogdahn, J Grassinger
    Cytokine, 2019-05-14;120(0):192-201.  2019-05-14
  16. Preeclampsia is Associated with Sex-Specific Transcriptional and Proteomic Changes in Fetal Erythroid Cells
    Authors: Z Masoumi, GE Maes, K Herten, Á Cortés-Cal, AG Alattar, E Hanson, L Erlandsson, E Mezey, M Magnusson, JR Vermeesch, M Familari, SR Hansson
    Int J Mol Sci, 2019-04-25;20(8):.  2019-04-25
  17. Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level
    Authors: J Chen, YR Kao, D Sun, TI Todorova, D Reynolds, SR Narayanaga, C Montagna, B Will, A Verma, AU Steidl
    Nat. Med., 2018-12-03;0(0):.  2018-12-03
  18. Human Intestinal Allografts Contain Functional Hematopoietic Stem and Progenitor Cells that Are Maintained by a Circulating Pool
    Authors: J Fu, J Zuber, M Martinez, B Shonts, A Obradovic, H Wang, SP Lau, A Xia, EE Waffarn, K Frangaj, TM Savage, MT Simpson, S Yang, XV Guo, M Miron, T Senda, K Rogers, A Rahman, SH Ho, Y Shen, A Griesemer, DL Farber, T Kato, M Sykes
    Cell Stem Cell, 2018-11-29;0(0):.  2018-11-29
  19. Targeting the ER-mitochondria interface sensitizes leukemia cells towards cytostatics
    Authors: F Koczian, O Nag?o, J Vomacka, B Vick, P Servatius, T Zisis, B Hettich, U Kazmaier, SA Sieber, I Jeremias, S Zahler, S Braig
    Haematologica, 2018-10-11;0(0):.  2018-10-11
  20. AMPK/FIS1-Mediated Mitophagy Is Required for Self-Renewal of Human AML Stem Cells
    Authors: S Pei, M Minhajuddi, B Adane, N Khan, BM Stevens, SC Mack, S Lai, JN Rich, A Inguva, KM Shannon, H Kim, AC Tan, JR Myers, JM Ashton, T Neff, DA Pollyea, CA Smith, CT Jordan
    Cell Stem Cell, 2018-06-14;0(0):.  2018-06-14
  21. Pharmacological inhibition of the transcription factor PU.1 in leukemia
    Authors: I Antony-Deb, A Paul, J Leite, K Mitchell, HM Kim, LA Carvajal, TI Todorova, K Huang, A Kumar, AA Farahat, B Bartholdy, SR Narayanaga, J Chen, A Ambesi-Imp, AA Ferrando, I Mantzaris, E Gavathioti, A Verma, B Will, DW Boykin, WD Wilson, GM Poon, U Steidl
    J. Clin. Invest., 2017-10-30;0(0):.  2017-10-30
  22. Proteomic analysis of JAK2V617F-induced changes identifies potential new combinatorial therapeutic approaches
    Authors: S Pearson, AJK Williamson, R Blance, TCP Somervaill, S Taylor, N Azadbakht, AD Whetton, A Pierce
    Leukemia, 2017-05-23;31(12):2717-2725.  2017-05-23
  23. Aerobic exercise in humans mobilizes HSC in an intensity dependent manner
    Authors: Gianni Parise
    J. Appl. Physiol., 2016-11-23;0(0):jap.00696.201.  2016-11-23
  24. Rational Design of a Parthenolide-based Drug Regimen that Selectively Eradicates Acute Myelogenous Leukemia Stem Cells
    J Biol Chem, 2016-08-29;0(0):.  2016-08-29
  25. PAK1 is a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome.
    Authors: Pandolfi A, Stanley R, Yu Y, Bartholdy B, Pendurti G, Gritsman K, Boultwood J, Chernoff J, Verma A, Steidl U
    Blood, 2015-07-13;126(9):1118-27.  2015-07-13
  26. miR-9 is a tumor suppressor in pediatric AML with t(8;21).
    Authors: Emmrich, S, Katsman-Kuipers, J E, Henke, K, Khatib, M E, Jammal, R, Engeland, F, Dasci, F, Zwaan, C M, den Boer, M L, Verboon, L, Stary, J, Baruchel, A, de Haas, V, Danen-van Oorschot, A A, Fornerod, M, Pieters, R, Reinhardt, D, Klusmann, J H, van den Heuvel-Eibrink, M M
    Leukemia, 2013-11-25;28(5):1022-32.  2013-11-25
  27. Activity of the hypoxia-activated prodrug, TH-302, in preclinical human acute myeloid leukemia models.
    Authors: Portwood S, Lal D, Hsu Y, Vargas R, Johnson M, Wetzler M, Hart C, Wang E
    Clin Cancer Res, 2013-10-02;19(23):6506-19.  2013-10-02
  28. Acellular bone marrow extracts significantly enhance engraftment levels of human hematopoietic stem cells in mouse xeno-transplantation models.
    Authors: Zibara K, Hamdan R, Dib L
    PLoS ONE, 2012-07-02;7(7):e40140.  2012-07-02
  29. Differential requirements for hematopoietic commitment between human and rhesus embryonic stem cells.
    Authors: Rajesh</LastName><ForeNam D</Initial, Rajesh D, Chinnasamy N, Mitalipov SM, Wolf DP, Slukvin I, Thomson JA, Shaaban AF
    Stem Cells, 2007-02-01;25(2):490-9.  2007-02-01

FAQs

  1. What is the difference between the Complete (Catalog # HSC003) and Enriched (Catalog # HSC005) Methylcellulose?

    • The Complete Methylcellulose Media (Catalog # HSC003) contains four growth factors (rhSCF, rhGM-CSF, rhIL-3, and rhEPO) whereas the Enriched Media (Catalog # HSC005) contains six growth factors (rhSCF, rhGM-CSF, rhIL-3, rhEPO, rhG-CSF, and rhIL-6) for hematpoietic stem cell (HSC) differentiation.

      HSC003 is typically used with an unpurified heterogeneous population of cells. PBMCs may be mixed with HSCs and the included growth factors will stimulate the PBMCs to express other growth factors to promote differentiation of HSCs.

      HSC005 is typically used with purified HSCs and it includes growth factors to differntiate HSCs without assistance from growth factors expressed from PBMCs.

  2. What are the differences between in Methylcellulose Complete Media with EPO (Catalog # HSC003) and without EPO (Catalog # HSC004)?
    • EPO is a potent cytokine used to stimulate differentiation of HSCs. EPO is needed for erythrocyte development. Media without EPO offers researchers the ability to expand and differentiate their HSCs and then to test supplemental drugs or compounds of interest.

  3. Can the CFU assay using Methycellulose based media be performed using frozen PBMCs instead of fresh PBMCs?

    • Yes, the CFU assay can be performed using frozen PBMCs.  The PBMCs can be frozen in DMEM containing 10% FBS and 10% DMSO.

  4. Why does the Human, Mouse and Rat colony forming assay protocol (CFC assay protocol) recommed use of non-tissue culture treated petri dishes?

    • The CFC assay promotes the growth of cells as colonies suspended in methylcellulose. However, if you use tissue culture treated dishes, the cells will also adhere and grow out on the bottom of the plate. Sometimes this appears as a round colony that is sticking and growing out on the edges (like an egg) and sometimes you can see patches of a monolayer. This makes it difficult to see the suspended colonies.

  5. Burst Forming Unit-Erythroid (BFU-E ) colonies representing erythorid progenitors appear to be low in frequency.  Is there a strategy to count these colonies and visualize them?

    • It is true that BFU-E colonies are low in frequency. To count and see good BFU-E colonies,  the CFC assay is set up at two cell densities.  For counting BFU-E colonies, a 10X cell concentration of  1.5-3x105 cells/mL  is used. For properly visualizing the BFU-E colonies,  an assay at half that cell density is used.

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