5.Determine the Cell Count.
a. Calculate the total cells counted in the four corner squares.
1) If the total cell count is less than 100, or if more than 10% of the cells counted appear to be clustered, carefully re-mix the original cell suspension and repeat steps 2 through 4 (above).
2) If the total cell count is greater than 400, dilute the suspension so the count will be 100-400 cells. Then repeat Steps 2 -4 (above).
NOTE: If satisfactory results are not achieved, contact your Clonetics® Technical Specialist by telephoning 800-852-5663.
b. Calculate the cell count using the equation: cells/ml = (n) x 104,
where: n = the average cell count per square of the four corner squares counted.
Example:If the calculated average (n) of cells in the four 1 mm corner squares of the hemacytometer is 30:
cells/ml = (n) x 104 (or) cells/ml = 30 x 10,000 = 300,000 cells/ml.
c. Determine the total number of cells in the total suspension volume.
1) Determine the total volume of the cell suspension.
2) Multiply the volume of the cell suspension by the "cells/ ml" value calculated above.
Example:If the initial suspension volume is 2 ml:
cells/ml x total volume = 300,000 cells/ml x 2 ml = 600,000 cells.
APPENDIX C
ASSESSMENT OF CELL VIABILITY WITH TRYPAN BLUE
Trypan blue is a dye that enables easy identification of dead cells. Dead cells take up the dye and appear blue with uneven cell membranes. By contrast, living cells repel the dye and appear refractile and colorless.
1. Prepare the hemacytometer for use.
a. Carefully clean all surfaces of the hemacytometer and cover slip.
b. Take care to ensure that all surfaces are completely dry using non-linting tissue.
c. Center the cover slip on the hemacytometer.
2. Transfer 50 ml of 0.4% Trypan Blue into a clean tube.
3. Add 50 ml of the prepared cell suspension into the tube containing the stain.
4. Mix the solution thoroughly, but gently. Take care to avoid making excessive bubbles.
5. Allow the mixture to sit for 2-3 minutes after mixing. (Do not let the cells sit in the dye for more than five minutes because both the living and dead cells will begin to take-up the dye after five minutes.)
6. Pipet approximately 9 microliters of the Trypan Blue/cell suspension mixture (this volume will vary with brand of hemacytometer) into one of the two counting chambers.
a. Use a clean pipet tip.
b. Be sure that the suspension is mixed thoroughly but gently before drawing the samples.
c. Fill the chambers slowly and steadily.
d. Avoid injecting bubbles into the chambers.
e. Do not overfill or underfill the chambers.
7. Determine Cell Viability.
a. Allow the suspension to settle in the chambers for at least 10 seconds.
b. Count all of the stained cells in each of the four corner squares of the hemacytometer.
c. Separately count all of the unstained cells in the same squares.
d. Calculate the cell viability using the equation:
% Cell Viability = number of unstained (living) cells / Total cells counted (stained + unstained) x 100%
Example: If a total of 300 cells (stained + unstained) are counted and 200 are identified as living cells (unstained), then the viability is calculated as:
% Cell viability =200 / 300 x 100% = 67%
IMPROVING CELL YIELD AND VIABILITY
Background
Several factors, or a combination of factors, contribute to low cell count and low cell viability. If cell yield or viability is unsatisfactory, use the following information to increase the success rate of future cultures.
Improving Cell Yield
If your cell yield is low (less than 50%), determine the cause(s) and possible solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).
Low Yield (Cell Count) | ||
CONDITION | POSSIBLE CAUSES | SOLUTIONS |
Majority of cells did not detach. |
|
|
95% of the cells detached but the yield was low. | Culture was under confluent at trypsinization. | Be sure to trypsinize at 70-90% confluence with at least 5 mitotic figures per field of view. |
If your cell viability is low (less than 50%), determine the possible cause(s) and solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).
Low Viability (Live Cells vs. Dead Cells) | ||
CONDITION | POSSIBLE CAUSES | SOLUTIONS |
Trypsin/EDTA damaged the cells. |
|
|
Culture vessel was too confluent. | Culture was too confluent at trypsinization. | Be sure to trypsinize at 70-90% confluence with about five mitotic figures per field of view. |
Cell growth slowed before 90% confluence and cells look dull and non- refractile. | The most probable cause is failure to increase the volume of medium used as the cell confluency increased. The cells become mildly starved and are not able to recover after trypsinization. | Change medium and increase volume as recommended. Please observe all guidelines. |
Once you have determined how to achieve high yield and high viability, subculture the remaining flasks.
2024年4月23日,北京理工大学生命学院肖振宇副教授、中国科学院动物研究所王红梅、于乐谦、郭靖涛研究员、中国农业大学魏育蕾教授、郑州大学第一附属医院何南南助理研究员在国际学术期刊Cell发表文章《3......
2019年10月3日,加州大学圣地亚哥分校BrendaL.Bloodgood团队(G.StefanoBrigidi为第一作者)在Cell在线发表题为“GenomicDecodingofNeuronal......
为迎合高速发展的半导体市场需求和逐渐增加的产品线,韩国ParkSystems于2023年9月15日在韩国果川市隆重举行了ParkSystems新总部大楼的奠基仪式。伴随着同期ParkSystems在韩......
美国宾夕法尼亚大学佩雷尔曼医学院科研人员发现,蚂蚁的血脑屏障在控制其行为方面起着积极的作用。血脑屏障可以调节蚂蚁大脑中的激素水平,从而影响他们在蚁群中的行为。相关研究成果发表在《Cell》杂志上。研究......
2023年9月18日,韩国ParkSystems再次隆重入选“福布斯亚洲2023年十亿美元以下最佳企业”榜单。此次入选标志着ParkSystems继2020年跻身福布斯200强之后,第二次荣登著名的福......
RNA引导系统利用引导RNA和靶核酸序列之间的互补性来识别遗传元件,在原核生物和真核生物的生物过程中都起着核心作用。例如,原核CRISPR-Cas系统为细菌和古细菌提供了对外来遗传因子的适应性免疫。C......
大约700万年前,人类从我们最接近的动物亲戚黑猩猩那里分离出来,在进化树上形成了我们自己的分支。在此后的时间里---从进化的角度看是短暂的---我们的祖先进化出了使我们成为人类的性状,包括比黑猩猩大得......
生命起源于一颗受精卵。精子“翻山越岭”遇见卵子的能力,是生命发生的必要条件。如果精子的运动能力出现异常,自然受孕的成功率便会大大降低;当精液中精子向前运动的比例低于32%时,则被定义为“弱精症(ast......
多细胞生物在发育过程中,存在着多种预定的、受到精确控制的细胞程序性死亡,例如细胞凋亡(Apoptosis)、程序性坏死(Necroptosis)、细胞焦亡(Pyroptosis),以及铁死亡(Ferr......
近日,国际学术期刊Cell子刊CellReports刊发了中国科学院海洋研究所在海洋动物细胞程序性死亡方面的最新研究成果。 皱纹盘鲍细胞焦亡激活通路及免疫调控示意图 海......