Thank you so much for the instructional video. One quick question in the "second wash of isolated PBMCs" section. The video mentions that "if concerned about platelets, run centrifuge at 200 x g for 10 min, with brake-off." 1) Why would reducing the centrifuge speed "protect" platelets? 2) In what circumstances should I be worried about platelets? In what assays/analysis are platelets used for (if there are any examples)?
Platelets, by nature, are very sticky, and when activated, will adhere easily to other sticky cell types (i.e. monocytes, eosinophils, etc.), creating cell clumps. The video is describing a method to remove platelets from the enriched mononuclear cells by performing one of the washes at 120 x g for 10 minutes at room temperature, with the brake off. This centrifugation will pellet mononuclear cells but not platelets, so they can be removed by aspirating the supernatant following the spin. Another method to reduce platelet contamination with SepMate™ is to pipette off some of the supernatant above the mononuclear cell layer before pouring to collect the mononuclear cells. Both methods are described in the Product Information Sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf ) for SepMate™. If you have any more questions, feel free to email us at techsupport@stemcell.com.
Acceleration rate refers to the increase in speed, while deceleration rate refers to the reduction in speed. In the context of centrifugation, deceleration rate settings are often called braking settings. Some centrifuges offer the capability to modify the acceleration and deceleration settings. For instance, the centrifuge used in this video allows you to choose acceleration settings from 1 (slow acceleration) to 9 (acceleration at maximum power) and deceleration settings from 0 (brake OFF) to 10 (deceleration/brake at maximum power). Using SepMate™ for PBMC isolation by density gradient centrifugation allows you to set the brake ON, reducing the time needed for the isolation of PBMCs compared to using regular tubes. Note that different makes and models of centrifuges may provide different rates of deceleration when braking, and with some models it may be necessary to reduce the deceleration rate to medium or low. If you have any more questions, feel free to email us at techsupport@stemcell.com.
In my case, I always end up with pellets contaminated with RBCs, even though I follow the procedure step by step according to the protocol. I was wondering if using a red blood cell lysis buffer could be of any help, and I found ammonium chloride in your catalog. At what stage of the process can I use it? ( i use sepmate for pbmc isolation). Thanks!
After centrifugation, some red blood cells (RBCs) may be present above the SepMate™ insert. These RBCs will not affect performance. RBCs can be more frequent in the PBMC fraction when dealing with older (24hr+) whole blood samples, and we recommend centrifuging the SepMate™ tubes for 20 minutes at 1200 x g (08:00 of the video). After centrifugation, the top layer can be poured off (10:45 of the video), washed once (11:40 of the video), and the cell pellet can be treated with Ammonium Chloride solution. If you have any more questions, feel free to email us at techsupport@stemcell.com.
If you set the brake on, your PBMC will get more RBC. If you pour it, you will get more RBC too. It's work for none sick blood only. My experience, If you put ficoll under the plastic insert with 15ml whole blood and dilute 15ml PBS, You will get good cell viability. SepMate tube will get more RBC than Direct Ficoll Method. If you done in 1 hour, you will get high cell viability.
Our SepMate tubes have been optimized on whole blood samples collected from healthy donors that have been diluted 1:1, with a hematocrit post-dilution of 15-28% (30-56% pre-dilution). The hematocrit or RBC content of the samples is important because the packed RBC volume is used to displace the density medium and PBMC layer up above the plastic insert in the tubes. If the RBC volume is too low, the enriched MNCs will get stuck below the insert and cannot be easily poured-off or retrieved. As noted in the product information sheet, some RBCs may be present on the surface of the SepMate insert after centrifugation. These RBCs will not affect performance. Also, for whole blood samples older than 24 hours, we do recommend spinning the tubes longer (20 minutes). In terms of pouring off the supernatant, one should do this in a quick, smooth and confident motion, and hold the tube upside down only for 1-2 seconds. You want to minimize the amount of time you hold the tube diagonally. If you have any further questions, please reach out to us by emailing techsupport@stemcell.com!
Hi Ashley, while we have not tried this in-house, some customers have successfully harvested plasma prior to using SepMate by centrifuging the undiluted whole blood (800 xg for 15-20 minutes, without density gradient media), collecting the plasma layer, and resuspending the pelleted cells in a volume of recommended medium (PBS + 2% FBS) equal to the the volume of plasma removed prior to starting the SepMate protocol. Please note that the SepMate protocol calls for diluting the blood 1:1 with recommended medium, and that this step should still be performed to avoid incomplete separation due to low liquid volume. If you have any more questions, feel free to contact us at techsupport@stemcell.com.
Hi Ashley, yes, the blood must be diluted with PBS and FBS regardless of the anticoagulant used. The SepMate isolation will not work properly if it is not diluted. If you have any more questions, feel free to email us at techsupport@stemcell.com!
To prevent PBMC cell clumping after processing fresh whole blood with SepMate tubes, make sure you are washing the pellet with PBS + 2%FBS. If you are proceeding with an EasySep kit after washing the PBMCs, make sure you resuspend the cells in a suitable volume of PBS containing 2% FBS and 1 mM EDTA, or EasySep buffer prior to starting the isolation. If you follow the protocol on the SepMate product information sheet and continue to have issues with PBMC clumping, feel free to reach out to our Product & Scientific Support team for further assistance (techsupport@stemcell.com).
Our recommended protocol for density gradient centrifugation (with or without SepMate™ tubes) includes two wash steps. Similarly, our standalone protocol for the RosetteSep™ Human Progenitor Cell Basic Pre-Enrichment Kit (Catalog #15226) lists two washes (1 x 300 x g and 1 x 120 x g). Protocols that require two washes apply to a wider variety of downstream applications that benefit from higher cell purity / reduced platelet contamination. Although the Erythroid Progenitor Reprogramming Kit (Catalog #05924) uses the same RosetteSep™ cocktail that is included in the RosetteSep™ Human Progenitor Cell Basic Pre-Enrichment Kit (Catalog #15226), the Erythroid Progenitor Reprogramming Kit protocol only requires a single 300 x g wash of the enriched hematopoietic progenitors. The 120 x g wash is intentionally excluded from the Erythroid Progenitor Reprogramming Kit protocol because it has an intermediate expansion step that increases the number of erythroid progenitors before starting the reprogramming step. The additional wash at 120 x g that is normally included to reduce platelet contamination would therefore not improve the performance of the downstream workflow in this particular application. Additionally, washes inherently result in some cell loss, so excluding the wash step helps retain more starting cells that can then be used for downstream reprogramming. If you have any more questions, feel free to email us at techsupport@stemcell.com.
When washing, are the centrifuges at room temperature (20 to 25°C) too? Does the gradient quantity have to be 15ml? If it is 20mL, does it change performance? What is the maximum amount of blood diluted in PBS that can be used per SepMate tube?
Hi Marcos, For the wash steps, centrifuging at 300 x g for 8 minutes at room temperature, with the brake on, is recommended. The volume of density gradient medium to add to the SepMate™ tube depends on the size of the tube (15 mL or 50 mL) and the blood sample volume. 15 mL is the volume required for the 50 mL tubes. This information is available in Table 1 of the product information sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf). The volume of density gradient medium has been optimized based on the position of the insert and using a different volume than recommended may affect the performance of the isolation. SepMate™-15 (cat #85420 www.stemcell.com/sepmate-15-ivd.html) is designed to process from 0.5 to 5 mL of whole blood (before dilution), and SepMate™-50 (cat #85450 www.stemcell.com/sepmate-50-ivd.html) is designed to process from 5 to 17 mL of whole blood (before dilution). This information is available in Table 1 of the product information sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf). If you have any more questions, feel free to email us at techsupport@stemcell.com.
Human B cells can be purified during standard density gradient centrifugation (e.g. peripheral blood mononuclear cell (PBMC) isolation using Lymphoprep™, Ficoll-Paque™, RosetteSep™ DM-L Density Medium or RosetteSep™ DM-M Density Medium) with the use of the RosetteSep™ Human B Cell Enrichment Cocktail (Catalog # 15024/15064) (www.stemcell.com/rosettesep-human-b-cell-enrichment-cocktail.html). RosetteSep™ is a fast and easy immunodensity procedure for the isolation of untouched cells directly from whole blood. By crosslinking unwanted cells to red blood cells (RBCs) present in the sample, target cells are purified during standard density gradient centrifugation. For additional B cell isolation methods including immunomagnetic isolation, please refer to this Human B Cell Isolation Product Selection guide (cdn.stemcell.com/media/files/brochure/Human_B_Cell_Isolation_Product_Selection.pdf?_ga=2.219503065.1312914144.1666602988-1504112607.1666216542&_gac=1.152509003.1666346489.Cj0KCQjwhsmaBhCvARIsAIbEbH4yFJrn2h4F6TQBDpLhmF5EEA93zzpmtNXkIcLWdcWN8eF2ljsgcjoaAhotEALw_wcB). If you have any further questions, feel free to email us at techsupport@stemcell.com.
I have used Sepmate with the Heparin tubes. The issue was when I spin herapined bloods, the buffy layer often stick together. What should I do to avoid this? Thanks
Hi Trung, we may need more information about the issue but we are happy to help you troubleshoot via email. Please email us at techsupport@stemcell.com.
We do not recommend umbilical cord blood as a good source of MSCs for many reasons, including that the cord has to be fresh, there is high variability among cords, and the frequency of MSCs in cord blood is low. For cord-derived MSCs, we recommend isolating from cord tissue (sometimes referred to as Wharton Jelly) using the explant method. Please refer to this video here: www.stemcell.com/technical-resources/isolation-of-umbilical-cord-mesenchymal-stromal-cells-using-explant-method.html If you really have to isolate MSCs from cord blood, some of our customers have tried this kit: www.stemcell.com/rosettesep-human-mesenchymal-stem-cell-enrichment-cocktail.html If you have any more questions, feel free to email us at techsupport@stemcell.com!
Excellent video, thanks a lot. after isolating the PBMCs, if I want to perform Co-culture with cancer cells for immune infiltration study, do you have a video like this. Finally, isolation of macrophages (M1 and M2 conditions)and lymphocytes (T cell subtypes) from PBMCs, do we need to do it separately or together? would love to hear your insights with video. Thanks once more
Unfortunately, we don't have a video or validated protocol to suggest for co-culture of PBMC with cancer cells; however, we believe that this will be a helpful reference for you: bit.ly/3cROtyV Additionally, T cells and subsets of T cells can be isolated from PBMC with EasySep™ immunomagnetic cell separation using positive or negative selection methods as described in this video: bit.ly/3gF5pLi Finally, M1 and M2 macrophage are typically differentiated from isolated and cultured peripheral blood monocytes. A workflow and products for this are outlined in the Brochure: bit.ly/3xCfaQp Feel free to email us at techsupport@stemcell.com if you have any further questions.
After initial centrifugation, if the fraction above the insert remains red, is it best to (1) continue with wash and RBC lysis? Or (2) pour into new sepmate and respin? I've noticed that if temp is off, or whole blood has been sitting for >6hrs, the separation isn't ideal.
Hi, based on your description, it is likely an incomplete separation due to low temperature and/or an old sample. Assuming the red above the insert is due to RBC contamination but not hemolysis, our suggestion would be: 1) Follow the protocol instruction listed here: cdn.stemcell.com/media/files/pis/10000003327-PIS_03.pdf and keep all reagents, the blood, and the centrifuge itself at room temperature before the centrifugation process. If you already performed the centrifugation at a non-ideal temperature then you can place the SepMate holding the sample back into the centrifuge along with the appropriate balance and do another 10 minutes of spin at the same speed at room temperature. 2) For older samples, a longer spin will help. Usually, 10 minutes longer should do the trick. As above, if you have already took the SepMate out of the centrifuge, then you can place it back in with appropriate balances and do another 10 minutes of spin. Please note, if the redness is from hemolysis (unlikely based on your description, but the redness will appear transparent like a fruit punch red Gatorade in case of hemolysis), then centrifugation wouldn't help. You may need to investigate the storage conditions/transportation conditions of the blood. If you have any more questions, feel free to email us at techsupport@stemcell.com.
@@STEMCELLTechnologies thanks for the reply! This is helpful. I will be sure to try these tips out next time I get an incomplete separation. It's refreshing to know that your company is actively monitoring this channel.
There is no need to coat the plate. This is a helpful reference on how to culture PBMCs: link.springer.com/protocol/10.1007%2F7651_2014_99 If you have any other questions, please feel free to email techsupport@stemcell.com and we'll be happy to help!
I heard you very briefly mention the 50-mL SepMates can only take 5-17 mL of blood. Is that right? I didn't see that on the website or instructions anywhere. We are working with large blood volumes.
As outlined in the product information sheet (PIS) for SepMate-50/SepMate-15, these tubes only come in two sizes: 15mL and 50mL. The maximum blood volume that can be processed with SepMate-50 is 17mL. This information is found on the PIS: Table 1 or under Notes: cdn.stemcell.com/media/files/pis/10000003788-PIS_05.pdf Please feel free to email techsupport@stemcell.com if you have any further questions and we'll be happy to help!
Perfect explanation, thanks a lot from Egypt
Thank you so much for the instructional video. One quick question in the "second wash of isolated PBMCs" section. The video mentions that "if concerned about platelets, run centrifuge at 200 x g for 10 min, with brake-off."
1) Why would reducing the centrifuge speed "protect" platelets?
2) In what circumstances should I be worried about platelets? In what assays/analysis are platelets used for (if there are any examples)?
Platelets, by nature, are very sticky, and when activated, will adhere easily to other sticky cell types (i.e. monocytes, eosinophils, etc.), creating cell clumps.
The video is describing a method to remove platelets from the enriched mononuclear cells by performing one of the washes at 120 x g for 10 minutes at room temperature, with the brake off. This centrifugation will pellet mononuclear cells but not platelets, so they can be removed by aspirating the supernatant following the spin. Another method to reduce platelet contamination with SepMate™ is to pipette off some of the supernatant above the mononuclear cell layer before pouring to collect the mononuclear cells. Both methods are described in the Product Information Sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf ) for SepMate™.
If you have any more questions, feel free to email us at techsupport@stemcell.com.
Made my life easier. Thank you!
Can you please explain the acceleration and deacceleration?
Acceleration rate refers to the increase in speed, while deceleration rate refers to the reduction in speed. In the context of centrifugation, deceleration rate settings are often called braking settings.
Some centrifuges offer the capability to modify the acceleration and deceleration settings. For instance, the centrifuge used in this video allows you to choose acceleration settings from 1 (slow acceleration) to 9 (acceleration at maximum power) and deceleration settings from 0 (brake OFF) to 10 (deceleration/brake at maximum power).
Using SepMate™ for PBMC isolation by density gradient centrifugation allows you to set the brake ON, reducing the time needed for the isolation of PBMCs compared to using regular tubes. Note that different makes and models of centrifuges may provide different rates of deceleration when braking, and with some models it may be necessary to reduce the deceleration rate to medium or low.
If you have any more questions, feel free to email us at techsupport@stemcell.com.
In my case, I always end up with pellets contaminated with RBCs, even though I follow the procedure step by step according to the protocol. I was wondering if using a red blood cell lysis buffer could be of any help, and I found ammonium chloride in your catalog. At what stage of the process can I use it? ( i use sepmate for pbmc isolation). Thanks!
After centrifugation, some red blood cells (RBCs) may be present above the SepMate™ insert. These RBCs will not affect performance. RBCs can be more frequent in the PBMC fraction when dealing with older (24hr+) whole blood samples, and we recommend centrifuging the SepMate™ tubes for 20 minutes at 1200 x g (08:00 of the video). After centrifugation, the top layer can be poured off (10:45 of the video), washed once (11:40 of the video), and the cell pellet can be treated with Ammonium Chloride solution.
If you have any more questions, feel free to email us at techsupport@stemcell.com.
If you set the brake on, your PBMC will get more RBC.
If you pour it, you will get more RBC too.
It's work for none sick blood only.
My experience, If you put ficoll under the plastic insert with 15ml whole blood and dilute 15ml PBS, You will get good cell viability.
SepMate tube will get more RBC than Direct Ficoll Method.
If you done in 1 hour, you will get high cell viability.
Our SepMate tubes have been optimized on whole blood samples collected from healthy donors that have been diluted 1:1, with a hematocrit post-dilution of 15-28% (30-56% pre-dilution). The hematocrit or RBC content of the samples is important because the packed RBC volume is used to displace the density medium and PBMC layer up above the plastic insert in the tubes. If the RBC volume is too low, the enriched MNCs will get stuck below the insert and cannot be easily poured-off or retrieved.
As noted in the product information sheet, some RBCs may be present on the surface of the SepMate insert after centrifugation. These RBCs will not affect performance. Also, for whole blood samples older than 24 hours, we do recommend spinning the tubes longer (20 minutes).
In terms of pouring off the supernatant, one should do this in a quick, smooth and confident motion, and hold the tube upside down only for 1-2 seconds. You want to minimize the amount of time you hold the tube diagonally.
If you have any further questions, please reach out to us by emailing techsupport@stemcell.com!
Thank you so much for the demo! We harvest the plasma... Would I still be able to do that if I were to use the SepMate? Thank you in advance!
Hi Ashley, while we have not tried this in-house, some customers have successfully harvested plasma prior to using SepMate by centrifuging the undiluted whole blood (800 xg for 15-20 minutes, without density gradient media), collecting the plasma layer, and resuspending the pelleted cells in a volume of recommended medium (PBS + 2% FBS) equal to the the volume of plasma removed prior to starting the SepMate protocol. Please note that the SepMate protocol calls for diluting the blood 1:1 with recommended medium, and that this step should still be performed to avoid incomplete separation due to low liquid volume. If you have any more questions, feel free to contact us at techsupport@stemcell.com.
If my whole blood is coming from an edta tube do I still need to dilute the wb with pbs 2% fbs? Thank you
Hi Ashley, yes, the blood must be diluted with PBS and FBS regardless of the anticoagulant used. The SepMate isolation will not work properly if it is not diluted. If you have any more questions, feel free to email us at techsupport@stemcell.com!
I have a question. Can I use the sepmate for producing in GMP facility ?
Hi, we would need more information. Please email us at techsupport@stemcell.com with your experimental setup.
Great demonstration! What do you recommend for clumping of the PBMCs during the centrifuge steps?
To prevent PBMC cell clumping after processing fresh whole blood with SepMate tubes, make sure you are washing the pellet with PBS + 2%FBS. If you are proceeding with an EasySep kit after washing the PBMCs, make sure you resuspend the cells in a suitable volume of PBS containing 2% FBS and 1 mM EDTA, or EasySep buffer prior to starting the isolation. If you follow the protocol on the SepMate product information sheet and continue to have issues with PBMC clumping, feel free to reach out to our Product & Scientific Support team for further assistance (techsupport@stemcell.com).
So when do I need to wash twice, such as when I need to amplify PBMCs and induce them into IPSCs in the future
i have read the 05924 protocol which only ask a once wash
Our recommended protocol for density gradient centrifugation (with or without SepMate™ tubes) includes two wash steps. Similarly, our standalone protocol for the RosetteSep™ Human Progenitor Cell Basic Pre-Enrichment Kit (Catalog #15226) lists two washes (1 x 300 x g and 1 x 120 x g). Protocols that require two washes apply to a wider variety of downstream applications that benefit from higher cell purity / reduced platelet contamination.
Although the Erythroid Progenitor Reprogramming Kit (Catalog #05924) uses the same RosetteSep™ cocktail that is included in the RosetteSep™ Human Progenitor Cell Basic Pre-Enrichment Kit (Catalog #15226), the Erythroid Progenitor Reprogramming Kit protocol only requires a single 300 x g wash of the enriched hematopoietic progenitors.
The 120 x g wash is intentionally excluded from the Erythroid Progenitor Reprogramming Kit protocol because it has an intermediate expansion step that increases the number of erythroid progenitors before starting the reprogramming step. The additional wash at 120 x g that is normally included to reduce platelet contamination would therefore not improve the performance of the downstream workflow in this particular application. Additionally, washes inherently result in some cell loss, so excluding the wash step helps retain more starting cells that can then be used for downstream reprogramming.
If you have any more questions, feel free to email us at techsupport@stemcell.com.
When washing, are the centrifuges at room temperature (20 to 25°C) too?
Does the gradient quantity have to be 15ml? If it is 20mL, does it change performance?
What is the maximum amount of blood diluted in PBS that can be used per SepMate tube?
Hi Marcos,
For the wash steps, centrifuging at 300 x g for 8 minutes at room temperature, with the brake on, is recommended.
The volume of density gradient medium to add to the SepMate™ tube depends on the size of the tube (15 mL or 50 mL) and the blood sample volume. 15 mL is the volume required for the 50 mL tubes. This information is available in Table 1 of the product information sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf). The volume of density gradient medium has been optimized based on the position of the insert and using a different volume than recommended may affect the performance of the isolation.
SepMate™-15 (cat #85420 www.stemcell.com/sepmate-15-ivd.html) is designed to process from 0.5 to 5 mL of whole blood (before dilution), and SepMate™-50 (cat #85450 www.stemcell.com/sepmate-50-ivd.html) is designed to process from 5 to 17 mL of whole blood (before dilution). This information is available in Table 1 of the product information sheet (cdn.stemcell.com/media/files/pis/10000003788-PIS_09.pdf).
If you have any more questions, feel free to email us at techsupport@stemcell.com.
i want lymphocytes, especially B- lymphocytes, which method could be best
Human B cells can be purified during standard density gradient centrifugation (e.g. peripheral blood mononuclear cell (PBMC) isolation using Lymphoprep™, Ficoll-Paque™, RosetteSep™ DM-L Density Medium or RosetteSep™ DM-M Density Medium) with the use of the RosetteSep™ Human B Cell Enrichment Cocktail (Catalog # 15024/15064) (www.stemcell.com/rosettesep-human-b-cell-enrichment-cocktail.html). RosetteSep™ is a fast and easy immunodensity procedure for the isolation of untouched cells directly from whole blood. By crosslinking unwanted cells to red blood cells (RBCs) present in the sample, target cells are purified during standard density gradient centrifugation. For additional B cell isolation methods including immunomagnetic isolation, please refer to this Human B Cell Isolation Product Selection guide (cdn.stemcell.com/media/files/brochure/Human_B_Cell_Isolation_Product_Selection.pdf?_ga=2.219503065.1312914144.1666602988-1504112607.1666216542&_gac=1.152509003.1666346489.Cj0KCQjwhsmaBhCvARIsAIbEbH4yFJrn2h4F6TQBDpLhmF5EEA93zzpmtNXkIcLWdcWN8eF2ljsgcjoaAhotEALw_wcB).
If you have any further questions, feel free to email us at techsupport@stemcell.com.
I have used Sepmate with the Heparin tubes. The issue was when I spin herapined bloods, the buffy layer often stick together. What should I do to avoid this? Thanks
Hi Trung, we may need more information about the issue but we are happy to help you troubleshoot via email. Please email us at techsupport@stemcell.com.
Is that protocol be used for isolation of MSCs from Umbilical Cord blood ......
We do not recommend umbilical cord blood as a good source of MSCs for many reasons, including that the cord has to be fresh, there is high variability among cords, and the frequency of MSCs in cord blood is low. For cord-derived MSCs, we recommend isolating from cord tissue (sometimes referred to as Wharton Jelly) using the explant method. Please refer to this video here: www.stemcell.com/technical-resources/isolation-of-umbilical-cord-mesenchymal-stromal-cells-using-explant-method.html
If you really have to isolate MSCs from cord blood, some of our customers have tried this kit: www.stemcell.com/rosettesep-human-mesenchymal-stem-cell-enrichment-cocktail.html
If you have any more questions, feel free to email us at techsupport@stemcell.com!
Excellent video, thanks a lot. after isolating the PBMCs, if I want to perform Co-culture with cancer cells for immune infiltration study, do you have a video like this. Finally, isolation of macrophages (M1 and M2 conditions)and lymphocytes (T cell subtypes) from PBMCs, do we need to do it separately or together? would love to hear your insights with video. Thanks once more
Unfortunately, we don't have a video or validated protocol to suggest for co-culture of PBMC with cancer cells; however, we believe that this will be a helpful reference for you: bit.ly/3cROtyV
Additionally, T cells and subsets of T cells can be isolated from PBMC with EasySep™ immunomagnetic cell separation using positive or negative selection methods as described in this video: bit.ly/3gF5pLi
Finally, M1 and M2 macrophage are typically differentiated from isolated and cultured peripheral blood monocytes. A workflow and products for this are outlined in the Brochure: bit.ly/3xCfaQp
Feel free to email us at techsupport@stemcell.com if you have any further questions.
After initial centrifugation, if the fraction above the insert remains red, is it best to (1) continue with wash and RBC lysis? Or (2) pour into new sepmate and respin?
I've noticed that if temp is off, or whole blood has been sitting for >6hrs, the separation isn't ideal.
Hi, based on your description, it is likely an incomplete separation due to low temperature and/or an old sample.
Assuming the red above the insert is due to RBC contamination but not hemolysis, our suggestion would be:
1) Follow the protocol instruction listed here: cdn.stemcell.com/media/files/pis/10000003327-PIS_03.pdf and keep all reagents, the blood, and the centrifuge itself at room temperature before the centrifugation process.
If you already performed the centrifugation at a non-ideal temperature then you can place the SepMate holding the sample back into the centrifuge along with the appropriate balance and do another 10 minutes of spin at the same speed at room temperature.
2) For older samples, a longer spin will help. Usually, 10 minutes longer should do the trick.
As above, if you have already took the SepMate out of the centrifuge, then you can place it back in with appropriate balances and do another 10 minutes of spin.
Please note, if the redness is from hemolysis (unlikely based on your description, but the redness will appear transparent like a fruit punch red Gatorade in case of hemolysis), then centrifugation wouldn't help. You may need to investigate the storage conditions/transportation conditions of the blood. If you have any more questions, feel free to email us at techsupport@stemcell.com.
@@STEMCELLTechnologies thanks for the reply! This is helpful. I will be sure to try these tips out next time I get an incomplete separation. It's refreshing to know that your company is actively monitoring this channel.
Lovely video. After isolating the PBMCs, do you plate then on coated plates or non plated plates are fine? because the cells grow as suspension
There is no need to coat the plate. This is a helpful reference on how to culture PBMCs: link.springer.com/protocol/10.1007%2F7651_2014_99
If you have any other questions, please feel free to email techsupport@stemcell.com and we'll be happy to help!
@@STEMCELLTechnologies thank you very much
I heard you very briefly mention the 50-mL SepMates can only take 5-17 mL of blood. Is that right? I didn't see that on the website or instructions anywhere. We are working with large blood volumes.
As outlined in the product information sheet (PIS) for SepMate-50/SepMate-15, these tubes only come in two sizes: 15mL and 50mL. The maximum blood volume that can be processed with SepMate-50 is 17mL. This information is found on the PIS: Table 1 or under Notes: cdn.stemcell.com/media/files/pis/10000003788-PIS_05.pdf
Please feel free to email techsupport@stemcell.com if you have any further questions and we'll be happy to help!
You can take up to 20ml of blood.