Navigation Links
The QIAGEN Guide to Animal Cell Culture

Part V: Counting cells

Welcome to the fifth of a series of articles providing useful hints for culturing animal cells. This article contains considerations for counting cells.

This protocol has been adapted from references 13. Protocols in this series are examples of methods for general cell culture and have not been rigorously validated and optimized by QIAGEN. There are many alternative protocols in current use.

Cell counting using a hemocytometer

It is often necessary to count cells, for example, when plating cells for transfection experiments. One method for counting cells is to use a hemocytometer. A hemocytometer contains 2 chambers (Figure 1). Each chamber is ruled into 9 major squares (volume of 0.1 mm3 or 1 x 104 ml each). Cell concentration is determined by counting the number of cells within a defined area of known depth (volume).

1. Clean the surface of the hemocytometer with 70% ethanol or another suitable disinfectant, taking care not to scratch the surface of the central area. Dry with lens paper.

2. Clean the coverslip, wet the edges very slightly, lay it over the grooves and central area of the hemocytometer and gently press down.

Tip - It is important that the coverslip is properly attached to obtain the correct chamber depth. The appearance of Newtons rings (bright and dark rings caused by interference in the air between the coverslip and the glass surface of the hemocytometer) will confirm that the coverslip is attached properly.

3. Harvest the cells, either by trypsinization (adherent cell cultures; see QIAGEN News 2002 No. 3, 20) or by centrifugation at 200 x g for 5 min (suspension cell cultures). Resuspend the cells in an appropriate volume of prewarmed growth medium. At least 106 cells/ml are required for accurate counting.

Tip - It may be necessary to centrifuge cells and resuspend in a smaller volume to obtain the desired cell concentration for counting. For adherent cells, it is important to produce a single-cell suspension after trypsinizing. Cell clumping will make counting difficult and inaccurate.

4. Mix the cell suspension sample thoroughly. Using a pipet, immediately transfer 20 l to the edge of one side of the coverslip to fill one chamber of the hemocytometer. Repeat for the second chamber.

Tip- The cell distribution should be homogeneous in both chambers.

Tip - The cell suspension is drawn under the coverslip and into the chamber by capillary action. The cell suspension should just fill the chamber. Blot off any surplus fluid without disturbing the sample underneath the coverslip.

5. Transfer the slide to the microscope, and view a large square ruled by 3 lines using a 10x objective and 10x ocular. Count the total number of cells in 5 of the 9 major squares.
Count cells that overlap the top and left border of squares but not those overlapping bottom and right borders (Figure 1). This prevents counting overlapping cells twice. If the cell density is too high, the cell suspension should be diluted, noting the dilution factor.

6. Repeat the counting for the second chamber to give a total of 10 squares.

7. Add the number of cells counted in all 10 squares together to give the number of cells in 1 x 103 ml. Multiply by 1000 to give the number of cells/ml.

IMPORTANT: If the original cell suspension was diluted for counting, multiply by the dilution factor to obtain the number of cells/ml.

8. Clean the hemocytometer and coverslip by rinsing with 70% ethanol and then with distilled water. Dry with lens paper.

Counting Cells Using a Hemocytometer Figure 1 A One chamber of a hemocytometer slide under 10x objective and 10x ocular. The chamber is divided into 9 major squares. B Detailed view of one of the 9 major squares. Only cells that overlap the top and left borders of squares should be counted to avoid overestimating the cell concentration. O: cells that should be counted; : cells that should be ignored.

References
1. Freshney, R.I. (1993) Culture of Animal Cells: a Manual of Basic Technique. 3rd ed. New York: Wiley-Liss.

2. Ausubel, F.M. et al., eds. (1991) Current Protocols in Molecular Biology. New York: Wiley Interscience.

3. Spector, D., Goldman, R.R., and Leinwand, L.A., eds. (1998) Cells: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. The QIAGEN Guide to Animal Cell Culture
2. The QIAGEN Guide to Animal Cell Culture
3. The QIAGEN Guide to Animal Cell Culture
4. The QIAGEN Guide to Animal Cell Culture
5. The QIAGEN Guide to Animal Cell Culture
6. QIAGEN Instrument Service insist on the best in service and support
7. QIAGEN Multiplex PCR Handbook
8. QIAGEN Multiplex PCR Kit
9. QIAGEN Plasmid Kits
10. Laser microdissection and nucleic acid purification - a Leica - QIAGEN cooperation
11. QIAGEN PCR CloningPlus Kit
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: The QIAGEN Guide Animal Cell Culture

(Date:6/2/2015)... , June 2, 2015 /CNW/ - Shell today celebrated ... in the premium category with an exhibit the size ... that included interactive demonstrations for media and the public, ... Shell V-Power NiTRO+ Premium Gasoline provides the best total ... engine performance: engine wear, gunk and corrosion. ...
(Date:6/2/2015)... SAN DIEGO and TORONTO ... APTO ; TSX: APS), a clinical-stage company developing ... today announced that the U.S. Food and Drug Administration ... APTO-253 for the treatment of acute myeloid leukemia (AML). ... the company,s lead product candidate in a Phase Ib ...
(Date:6/2/2015)... MIAMI (PRWEB) June 02, 2015 Global ... Latin America distributor for Bioquark, Inc.’s biologic products for the ... is the latest in Global Stem Cells Group’s ambitious expansion ... region’s growing demand for access to regenerative medicine and stem ... on the development of biologics that have the ability to ...
(Date:6/2/2015)... 2015 Research and Markets ( http://www.researchandmarkets.com/research/6wjpd6/bioburden_testing ) ... Market by Product , Test, Application & End User ... The bioburden testing market is expected to ... 2014, growing at a CAGR of 9.8% from 2014 ... microbial limit testing market is segmented into consumables and ...
Breaking Biology Technology:New Shell V-Power NiTRO+ Premium Gasoline Introduces Breakthrough Formulation for BEST TOTAL Engine Protection 2New Shell V-Power NiTRO+ Premium Gasoline Introduces Breakthrough Formulation for BEST TOTAL Engine Protection 3New Shell V-Power NiTRO+ Premium Gasoline Introduces Breakthrough Formulation for BEST TOTAL Engine Protection 4Aptose Biosciences Granted Orphan Drug Designation by the U.S. FDA for APTO-253 in Acute Myeloid Leukemia 2Aptose Biosciences Granted Orphan Drug Designation by the U.S. FDA for APTO-253 in Acute Myeloid Leukemia 3Aptose Biosciences Granted Orphan Drug Designation by the U.S. FDA for APTO-253 in Acute Myeloid Leukemia 4Adimarket Named Latin American Distributor for Bioquark Biologic Products for the Regeneration and Repair of Human Organs and Tissue with Stem Cells 2Adimarket Named Latin American Distributor for Bioquark Biologic Products for the Regeneration and Repair of Human Organs and Tissue with Stem Cells 3Bioburden Testing Market 2015 - Global Forecasts to 2019 2
... , , Alberta Centre ... health & medical seminar, , EDMONTON, Dec. 4 /PRNewswire/ ... how technologies like nanotechnology, biomaterials and microfluidics can play a ... help promote health and improve the quality, cost and outcomes ...
... , DALLAS, Dec. 3 ... Bulletin Board: ACCP), today provided an update on its European ... mucositis, a debilitating side effect of radiation treatment and chemotherapy. ... European countries, including the UK, Germany, Italy, Norway, Greece and ...
... , HOPKINTON, Mass., Dec. 3 Caliper Life Sciences, ... and services for drug discovery and life sciences research, today ... systems. Living Image software drives the more than 800 Caliper ... features advanced spectral unmixing tools to enhance fluorescent sensitivity and ...
Cached Biology Technology:Alberta companies delivering new products to the health & medical marketplace 2Access Pharmaceuticals Provides Update on MuGard Commercial Launch in Europe 2Access Pharmaceuticals Provides Update on MuGard Commercial Launch in Europe 3Access Pharmaceuticals Provides Update on MuGard Commercial Launch in Europe 4Caliper Life Sciences Launches Living Image(R) 4.0 Software 2
(Date:5/22/2015)... 22, 2015  NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" ... be ending its promotional pricing and making the Wocket generally ... Laurence Savin , Head of Marketing said, "We,ve extended the ... the end of May. To get your wocket at this ... open the store on June 1, you,ll receive a unique ...
(Date:5/21/2015)... The Sync Project™ , a global collaboration harnessing ... a collaborative partnership with Berklee,s Institute for Creative ... on original research, joint course development and an internship ... Sync Project and BerkleeICE are exploring the launch of ... academic year.  The course will focus ...
(Date:5/19/2015)... 2015 Fingerprint Cards (FPC) has received ... FPC1155 and FPC1035 from one if its module partners. Deliveries ... Q3 2015 and the sensors will be used by smartphone ... for 2015 hereby amount to 740 MSEK to date. In ... MSEK and a number of smaller orders not separately communicated ...
Breaking Biology News(10 mins):NXT-ID, Inc Announces Retail Price For Wocket Smart Wallet And Extends Discounted Promotional Price Till End Of May 2NXT-ID, Inc Announces Retail Price For Wocket Smart Wallet And Extends Discounted Promotional Price Till End Of May 3The Sync Project Announces Partnership with Berklee College of Music's Institute for Creative Entrepreneurship 2The Sync Project Announces Partnership with Berklee College of Music's Institute for Creative Entrepreneurship 3Fingerprint Cards Receives Touch Fingerprint Sensor Order of SEK 230 Million and Raises Revenue Guidance for 2015 2
... early stages of cancer could one day result from new ... Universitys School of Medicine has just published a study on ... which can play a crucial part in the onset of ... by gradually becoming shorter they can tell cells when it ...
... beleaguered growers of popular cucurbit crops like cucumbers and ... (Lagenaria siceraria) appear to have resistance to Zucchini yellow ... includes pumpkins, squashes and other kinds of melons, including ... in other parts of world, and is a particular ...
... 2, 2007The Keck School of Medicine of the University of ... the Kenneth T. & Eileen L. Norris Foundation that will ... centers devoted to the study of epigenetics. The grant ... field that holds great promise in the treatment and prevention ...
Cached Biology News:Research points towards early cancer detection 2Battling virus disease of watermelon with bottlegourds 2$10 million gift to support cutting-edge epigenome center at USC 2$10 million gift to support cutting-edge epigenome center at USC 3
... TProfessional Thermocycler combines top performance with exquisite ... high speed and unparalleled temperature uniformity. The ... programming in a spreadsheet or alternatively in ... Lid opens on the push of a ...
... Ultra Agarose Spin Kit is an advanced system for ... purified DNA can be used directly for any downstream ... DNA is ready to use in 56 minutes ... used for TAE and TBE gels No specialised ...
... been designed for recovery of DNA from TAE and ... Ultra fast protocol: DNA is ready to use ... to resuspend Bead uniformity prevents shearing of high ... PCR) DNA is eluted into sterile water or ...
... agarose gels The SpinPrep Gel DNA Kit enables ... > 12,000 bp in size from agarose gels. The ... followed by adsorption of the DNA to a silica ... step, the purified DNA is eluted in low-salt buffer. ...
Biology Products: