Enriching Rare Populations of Human CD34 Cells by FACS

February 10, 2021

ID6190

To CiteDCA Citation Guide

00:03Title of this training is enriching.
00:05Rare populations of primary
00:06human CD 34 positive cells
00:08by fax and has been prepared by members
00:10of the Kraus lab at Yale School of
00:13Medicine in partial fulfillment of
00:15the educational aims outlined in
00:17the mission of the Yale Cooperative
00:19Center for Excellence in Hematology.
00:21The purpose of this protocol is to analyze
00:25an isolate where populations of CD 34,
00:27positive matter, poetic stem,
00:29and progenitor cells from human G,
00:31CSF, mobilized peripheral blood,
00:33but maybe more broadly applicable
00:35to other starting cell populations.
00:38We will demonstrate the sorting
00:40strategy for human megakaryocytic
00:42over three progenitors in this video
00:44as an example of a rare population
00:46within the CD 34 positive fraction.
00:50Human CD 34 positive cells are
00:53a heterogeneous mixture of stem
00:54and progenitor cells at various
00:57stages of differentiation.
00:58To study a specific population of
01:01cells like high metabolic stem cells,
01:03this population must be enriched prior
01:06to any assets that may take place.
01:10Each progenitor population is
01:12defined by a combination of
01:14surface marker expression profiles.
01:17Fluorescence activated cell sorting
01:19or fax uses fluorescent markers to
01:22detect and separate the cells with
01:25desired surface marker expression.
01:27Building a customized flow cytometry panel.
01:31Designing a multi color panel for flow
01:35cytometry takes advanced preparation.
01:37Factors that need to be considered include.
01:41Configuration of the instrument.
01:43Such as the lasers and filters.
01:45It is equipped with. The number of
01:50colors or antibodies to be used.
01:53And the abundance of the antigens.
01:57We highly recommend utilizing Flora Finder as
02:01a platform for designing a multi color panel.
02:05Floor Finder allows the user to enter the
02:07exact specifications of their cytometer,
02:09including which lasers and filter
02:11sets are installed in the machine.
02:14Next,
02:14users can search and select which
02:17cell surface antigens and or
02:19fluorescent proteins they intend
02:21to analyze their cell sample for.
02:23It is useful to have prior knowledge of
02:26the antigen abundance on the cells in
02:28your sample and cell type of interest,
02:30but it is not necessary.
02:32Based on the specifications of
02:34the CYTOMETER and antigens to be
02:37detected that the user entered,
02:39Flora Finder populates a chart of
02:41commercially available antibodies
02:42for those antigens categorized by
02:44fluorescent channel such that the
02:47selection of 1 antibody in a particular
02:49channel automatically blocks the
02:51user from selecting additional
02:52antibodies in the same channel,
02:54thus reducing spectral overlap.
02:57Titrating staining antibodies.
02:59To ensure appropriate staining but
03:01not compromise antibody specificity,
03:03it is recommended that you titrate
03:06your antibodies prior to using
03:09them in a flow analysis or sort.
03:12To accomplish this utiliza cell
03:14sample that contains a mixture of
03:16cells that are positive for the
03:18antigen and negative pipette.
03:19A constant number of cells in a constant
03:23volume of staining solution into at
03:25least three but preferably 5 fax chips.
03:29Keep one tube as the unstained
03:31control and add increasing volumes
03:33of the antibody wish to titer to
03:36each subsequent tube.
03:37The range of volumes you choose
03:39should be based on the recommended
03:42dilution from the manufacturer.
03:44For example.
03:45If the recommended volume test for
03:48the manufacturer is 5 microliters,
03:50pipette 5 microliters of antibody
03:53into one tube.
03:55Two microliters of antibody into
03:57the next tube.
03:59One microliter of antibody into
04:01the next tube.
04:02And half a microliter of antibody
04:05into the last tube.
04:07At the Cytometer record,
04:09at least 10,000 events for each tube.
04:12Calculate the staining index by
04:14measuring the MFA of the negative cells
04:17and the standard deviation as well
04:19as the MFA for the positive cells.
04:22With these three measurements,
04:24calculate the staining index
04:26by subtracting the MFA of the
04:28negative population from the MFA
04:30of the positive population.
04:32And divide this by two times
04:34the standard deviation of
04:35the MFA of the negative population.
04:37Plot the staining index for each
04:39tube on a graph to identify the best
04:43dilution for that particular antibody.
04:45Sawing and staining CD. 34 positive cells.
04:51For the purposes of this didactic video,
04:54we will demonstrate the staining protocol for
04:57human megakaryocytic erythroid progenitors.
04:59You will need to substitute
05:00your antibodies of interest,
05:02but the protocol remains the same.
05:05With stained with CD,
05:0634 positive cells with a panel of
05:08seven antibodies, one of which is
05:11a cocktail of Lenny edge markers.
05:14To reduce the number of channels required for
05:16the sort and thus reduce spectral overlap,
05:19we use antibodies against the lineages.
05:21Markers that are directly
05:23conjugated to biotin.
05:25So we incorporate a secondary
05:26stain with streptavidin antibody
05:28directly conjugated to a floor.
05:30For that has limited spectral overlap
05:32with the rest of our staining panel.
05:37Required reagents include FBZ.
05:41Buffer one. Fax buffer.
05:45And titrated antibodies.
05:51Prepare labeled facts tubes
05:53for each compensation control.
05:55Fluorescence minus one control.
05:57As well as the sample tube.
06:03Compensation controls are cells
06:05stained with a single antibody.
06:08Compensation controls are required for
06:10every floor floor and allow the cytometry
06:13software to calculate and subtract
06:16spectral overlap between flora force.
06:18Fluorescence minus one controls
06:20are cells stained with all but one
06:22of the antibodies in the panel.
06:24It identifies the true negative of a floor
06:28for considering all the interference
06:30from the other staining floor force.
06:32These controls help determine the
06:35location of gates that separates cells not
06:38expressing the antigen from cells that are.
06:40They are required if the expression
06:43pattern of the antigen in your
06:45starting population is a continuum
06:48of negative to high expressing.
06:50Or the expression of the antigen is very low,
06:53so the positive signal is just
06:54slightly higher than the background
06:56fluorescence of the negative population.
07:00We quickly thaw cryopreserved CD
07:0234 positive cells in a 37 degree
07:05water bath until just a few ice
07:07crystals are left in the cryotube.
07:10Take care not to submerge the
07:12O-ring and cap of the cryo vial
07:14to reduce risk of contamination.
07:16When the cells are nearly thought,
07:18spray the cry of I'll with 70%
07:20ethanol and wipe it dry before
07:22bringing it into the hood to open.
07:25Be sure to practice strict
07:27aseptic technique in a bio safety
07:29cabinet rated for BSL 2 for the
07:31duration of this protocol.
07:41Add one milliliter of 100%
07:43FBS to a 50 mil conical tube.
07:51Add one milliliter of 100% FBS to the
07:54cryotube and gently transfer the full
07:56contents of the cryotube into the
07:5850 mil conical tube containing FBS.
08:13Add up to 50 milliliters of buffer.
08:16One dropwise cell suspension
08:17in the 50 mil conical tube.
08:19To delete out the DMSO in the freezing media.
08:25Spin at 1200 RPM 4 degrees
08:28Celsius for 10 minutes.
08:33When the spin is done,
08:35carefully discard the supernatant.
08:40Resuspend the pellet with 20
08:43milliliters of fax buffer.
08:44Take a representative 10 microliter
08:46aliquot for cell counting and repeat the
08:49spin of the conical tube at 1200 RPM,
08:514 degrees Celsius for 10 minutes.
08:55Add 10 microliters of two extra pen blue
08:58to the aliquot of cells and pipette to mix.
09:02Calculate the viable cell number
09:03in the sample control tubes.
09:05Each require 5 * 10 to the four cells.
09:09FMO tubes require as few as
09:112 * 10 to the 4th cells.
09:15When the second spin is complete,
09:17resuspend the pellet in 500
09:19microliters of Facs buffer an
09:20aliquot the necessary volume of
09:22cells required for control tubes.
09:24Add additional facts buffer to
09:26bring the sample tube volume back
09:28up to 500 microliters.
09:29Take care not to exceed a cell
09:32concentration of 20 * 10 to the six
09:34cells per milliliter in the sample tube.
09:39Aliquot the titrated amount
09:41of primary staining antibodies
09:43into the corresponding tubes.
09:45Be especially careful when allocating
09:47antibodies in the FMO tube so
09:49you don't accidentally add the
09:51antibody that should be absent.
09:53Additionally, it is important to
09:55note that the volume of antibodies
09:57added cannot exceed the maximum
09:59standing volume, which is.
10:00100 microliters for controls
10:02and one milliliter for sample.
10:05In other words,
10:06if you allocated 50 microliters of
10:08control cells to the control tubes,
10:10the volume of staining antibody
10:12cannot exceed 50 microliters so
10:14that the total staining volume
10:17remains 100 microliters.
10:18If the volume of the staining
10:20antibodies is less than 50 microliters,
10:22then add additional facts buffer to
10:24bring the final staining volume to
10:26100 microliters for control tubes and
10:28one milliliter for the sample tube.
10:33Once all cells Facs buffer an,
10:36antibodies are added to the staining tubes,
10:39incubate according to the
10:41manufacturer's recommendations.
10:42Most antibodies require incubation
10:44for 30 minutes on ice in the dark.
10:49After the primary stain is complete,
10:51add one milliliter of fax buffer
10:53to the control tubes and two
10:56milliliters of fax buffer to the
10:58sample tube and spin at 1200 RPM,
11:004 degrees Celsius for 10 minutes.
11:06When the spin is complete,
11:08discard the supernatant, taking
11:09great care not to discard the cell
11:11pellets at the bottom of the tubes.
11:22If any of the antibodies used in the
11:24first stain were conjugated to biotin,
11:26a secondary stain with
11:28streptavidin conjugated to a
11:29unique floor 4 will be required.
11:31Similarly to the primary stain,
11:33add the appropriate amount of
11:35streptavidin according to the
11:37titration to the appropriate tubes.
11:39You don't have to add this to single
11:42color control tubes that were not
11:44stained with a biotin conjugated
11:46antibody or to an FMO tube in which the
11:49biotin conjugated antibody was omitted.
11:51Bring the total staining volume
11:53up to 100 microliters for control
11:56teams or one milliliter for sample
11:58tubes with fax buffer.
12:00Incubate according to the
12:02manufacturer's recommendation.
12:04Most antibodies require incubation
12:05for 30 minutes on ice in the dark.
12:12Once secondary staining is complete,
12:14repeat the wash and spin steps as
12:16was done after the primary stain.
12:19Resuspend the control pellets in
12:21200 microliters of Facs buffer
12:23and 300 microliters of Facs
12:24buffer for the sample palette.
12:26Filter these cell suspensions through
12:28a 40 or 100 Micron filter depending
12:30on the expected size of yourselves
12:32and proceed to the cytometer.
12:41Prime just sorting the cells.
12:42Prepare collection tubes or plates
12:44to receive the sorted cells.
12:46Confirmed that Uber plate you wish
12:48to use fits the specification
12:50of the cytometer to be used.
12:52Typically, growth media supplemented
12:54with survival cytokines can be used
12:57to collect certain cells into and
12:59results in better post sort viability.
13:02Of special note,
13:03be sure to follow all safety policies
13:05and procedures when transporting
13:06yourselves to the cytometer.
13:08For example,
13:09unfixed human CD 34 positive cells
13:11are required to be transported
13:13in a secondary container that is
13:15labeled with a BSL two sticker.
13:19Once at the cytometer,
13:21set the color parameters that
13:23will be used to sort the cells.
13:25Create compensation tubes for each
13:27single color control tube you have
13:29and create a new specimen entitled,
13:31FMO and generate tubes for each FML you have.
13:35Also create a tube for your sample.
13:39Using the unstained control cells,
13:41adjust the voltages for each channel,
13:43including FC and SC,
13:44so that your cells are mostly clustered
13:47in the center of the FC SC dot plot
13:50and the background signal in each
13:52color channel is well below 2:50.
13:56Before recording,
13:56check those voltages on each of
13:58the single color control tubes
14:00as well as the sample tube.
14:01Adjust voltages for each channel
14:03so that the negative peak is below
14:0510 to the third and the positive
14:07peak is below 10 to the five.
14:11Once you are satisfied with the voltages,
14:13begin by recording single color
14:15controls and calculate the compensation.
14:17Once all have been recorded.
14:20Then record the FML's.
14:22Use the recorded data from the
14:24Fmo's to help draw and position
14:26gates based on true negatives.
14:30Once all of the gates of the
14:32sorting strategy have been drawn,
14:33load the sample onto the cytometer and
14:36set the population you wish to sort.
14:38Also specify what kind of
14:40collection container you're
14:41using and how many cells to sort.
14:44Then begin sorting your sample
14:45tube and collecting your enriched
14:47population for downstream applications.
15:01After the service complete, use the
15:03cells for your downstream application.
15:06If sorted cells were collected in a fax tube,
15:09add 2 milliliters of I MDM media
15:11to wash the cells in the sorted
15:13tube and spent at 1200 RPM,
15:154 degrees Celsius for 10 minutes.
15:21Resuspend the cell pellet in the
15:23appropriate growth media or freezing media.
15:28This concludes the protocol for
15:31Multicolor Facs sorting of CD 34.
15:34Positive amount Aquatic Center gender cells.