Dear all, I am working with bovine granulosa cells. Recently, I have moved to a new lab and I lost my previous protocol to prepare RBC lysis buffer which is suitable for granulosa cells collected for culturing. If anyone have a RBC lysis buffer recipe which is suitable for granulosa cells, please let me know.
Thanks in advance for your time.
I have 2 suggestions that I have used for human and rodent granulosa cells.
1. Gravity sedimentation and repeated dilution with PBS. Add a large volume of PBS (around 30ml to the sample in a 60ml Falcon tube. Leave to stand for 5 minutes. The more dense granulosa cells will sink to the bottom of the tube. Carefully aspirate most of the supernatant and then repeat 2 or 3 times. This will get rid of most of the RBCs.
2. Pellet the sample by centrifugation at 200-300g. Add 10 ml sterile water, mix rapidly (5-10 seconds) , then quickly add an equal volume of a 2x strength cell culture medium (available from Gibco) to restore the osmotic balance.. The RBCs are very sensitive to the hypo-osmotic shock and will rupture.
I hope this helps
Chris
This question was asked before on RG and answered by Isabelle - Müller (copy of answer is here)
Isabelle - Müller · 22.08 · 41.39 · Universität des Saarlandes
We use the following solution to lyse the RBCs:
ACK-Lysepuffer/RBC Lysis buffer (Erythrocytenlyse)
155mM NH4 Cl
10mM KHCO3
0,1mM EDTA
à pH 7,3
à steril filtration (0,2µM)
Depending on RBC concentration we add1-5 ml Lysis solution to the cell pellet, resuspend it and incubate for 1-10 min. That works very fine.
I used before
A10492 - ACK Lysing Buffer from Life Technologies, worked ok too
thank you so much for your suggestions @Subhash and @Ali Saeed
I have 2 suggestions that I have used for human and rodent granulosa cells.
1. Gravity sedimentation and repeated dilution with PBS. Add a large volume of PBS (around 30ml to the sample in a 60ml Falcon tube. Leave to stand for 5 minutes. The more dense granulosa cells will sink to the bottom of the tube. Carefully aspirate most of the supernatant and then repeat 2 or 3 times. This will get rid of most of the RBCs.
2. Pellet the sample by centrifugation at 200-300g. Add 10 ml sterile water, mix rapidly (5-10 seconds) , then quickly add an equal volume of a 2x strength cell culture medium (available from Gibco) to restore the osmotic balance.. The RBCs are very sensitive to the hypo-osmotic shock and will rupture.
I hope this helps
Chris
Use ACK lysis buffer ( look for any company protocol ) or deionized water to lyse RBC.
If needed, knock me at [email protected]
Hi Dhia Hussain,
Thanks for you suggestion. Currently I am using a combination of Ammonium chloride and Potassium chloride solution.
Hi Sohel
The RBC lysis buffer can be purchased from Sigma-Aldrichr or else can be prepared (8.3 gm/l NH4Cl in 0.01 M Tris–HCl buffer of pH 7.5) (Sontakke et al. Reproduction 2014). I used it for bovine GCs.
Good luck..
- Badges
- Science method